Affinity medicant conjugate

ABSTRACT

In an embodiment of the invention, a composition for treating a cell population comprises an Affinity Medicant Conjugate (AMC). The medicant moiety can be a toxin including an acylfulvene or a drug moiety. The affinity moiety can be an antibody, a binding protein, a steroid, a lipid, a growth factor, a protein, a peptide or non peptidic. The affinity moiety can be covalently bound to the medicant via a linker. Novel linkers that can be directed to cysteine, arginine or lysine residues based on solution pH allow greater flexibility in preserving and/or generating specific epitopes in the AMC.

PRIORITY CLAIM

This application is a divisional of and claims priority to (1) U.S.patent application Ser. No. 17/035,529 entitled “AFFINITY MEDICANTCONJUGATES”, inventor: Michael J. Kelner, filed Sep. 28, 2020, which isa continuation in part of (2) U.S. patent application Ser. No.15/986,727 entitled “AFFINITY MEDICANT CONJUGATES”, inventor: Michael J.Kelner, filed May 22, 2018 which is a continuation of (3) U.S. patentapplication Ser. No. 15/201,301 entitled “AFFINITY MEDICANT CONJUGATES”,inventor: Michael J. Kelner, filed Jul. 1, 2016, which issued as U.S.Pat. No. 9,980,926 on May 29, 2018 which is a continuation of (4) U.S.patent application Ser. No. 14/684,218 entitled “AFFINITY MEDICANTCONJUGATES”, inventor: Michael J. Kelner, filed Apr. 10, 2015, whichissued as U.S. Pat. No. 9,381,178 on Jul. 5, 2016 and which claimspriority to (5) the U.S. Provisional Application No. 61/978,195 entitled“AFFINITY MEDICANT CONJUGATES”, inventor: Michael J. Kelner filed Apr.10, 2014, which applications (1)-(5) are herein expressly incorporatedby reference in their entireties and for all purposes.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file MKEL-01047US7_ST25.TXT, createdSep. 7, 2021, 845,819 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to compositions and methods for treatingtarget molecules including cell populations with an affinity medicantconjugate such as an antibody drug conjugate.

BACKGROUND

The present invention is directed to Affinity Medicant Conjugates (AMC)including acylfulvene, Illudin and Syn-Illudin based conjugates,Affinity Medicant Linker Conjugates (AMLC), antibody-drug conjugates(ADC) and medicant-linker (ML) compounds, as well as to compositions ofthe same, and to methods for their use in treating cancer, an autoimmuneto methods of using Ligand Linker Medicant (LLM) conjugates and MLcompounds in vitro, in situ, and in vivo for the detection, diagnosis ortreatment of cells and associated pathological conditions.

SUMMARY OF INVENTION

There exists a continuing need for delivery of chemotherapeutic agentsfor which tumors do not have a medicant resistant phenotype and whichinhibit tumor growth, especially solid tumor growth, and which have anadequate therapeutic index to be effective for in vivo treatment. Theantibody medicant conjugates of the present invention can have utilityin a wide range of therapeutic applications in humans as well as inanimals in general. For example, such therapeutic applications caninclude: cancer, adenocarcinoma, carcinoma, breast cancer, prostatecancer, ovarian cancer, endometrial cancer, neuroendocrine tumors,infertility, polycystic ovary syndrome, endometriosis, and precociouspuberty. For example, veterinary and agricultural applications caninclude treatment of cancer, adenocarcinoma, carcinoma, ovarian cancer,endometrial cancer, neuroendocrine tumors, and endometriosis in farmyardand/or companion animals.

The methods of this invention include administration of an effectiveamount of an antibody medicant conjugate, preferably in the form of apharmaceutical composition, to an animal in need thereof. In a furtherembodiment, pharmaceutical compositions are disclosed containing anantibody medicant conjugate of the present invention in combination witha pharmaceutically acceptable carrier.

In various embodiments of the present invention, an affinity medicantconjugate is made up of an antibody 1110 linked to an illudin1 moiety1301. Various embodiments of the invention, are directed to the methodsfor the preparation, use, and to pharmaceutical compositions containingan illudin1 moiety 1301 linked to an antibody 1110 to form an antibodymedicant conjugate (AMC). In various embodiments the compounds of thepresent invention, the AMC can have the general formula shown in FIG.3A, where the antibody 1110 is bound to a linker 1200 which is bound toan illudin1 moiety 1301. In other various embodiments of the presentinvention, the compounds of the present AMC invention can have thegeneral formula shown in FIG. 3B, where a growth factor 1120 is bound toa linker 1200 which is bound to an illudin1 moiety 1301. In variousembodiments the compounds of the present invention includestereoisomers, solvates, and pharmaceutically acceptable salts thereof,where the linker 1200 is as defined in Table X, and the illudin1 1301 isas defined below in Table XI.

In various embodiment of the present invention, an antibody linked to anacylfulvene moiety acts as a ligand for an Epidermal Growth Factor (EGF)receptor (EGF-R) (SEQ. ID. 143) and directs the acylfulvene to cellpopulations expressing the EGF-R. These compounds are useful as a meansof treating cell populations expressing the EGF-R. In an embodiment ofthe present invention, these compounds are useful in treatment of tumorsin which the EGF-R is over expressed. In an embodiment of the presentinvention, these compounds are useful in treatment of cells in which theEGF-R acts as a marker. In various embodiment of the present invention,these compounds are useful in agricultural applications in which theEGF-R acts as a marker of cell populations involved in agriculturalproduction. In various embodiment of the present invention, thesecompounds are useful in veterinary medicine in which the EGF-R acts as amarker of cell populations involved in pet reproduction. In variousembodiments of the present invention, pharmaceutical compositionscomprising these compounds are used in the treatment of tumors in whichthe EGF-R is involved. In various embodiments of the present invention,methods of using the pharmaceutical compositions comprise thesecompounds to treat tumors in which the GH-R is involved.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention is described with respect to specific embodimentsthereof. Additional features can be appreciated from the Figures inwhich:

FIG. 1 shows a schematic description of the Affinity Medicant Conjugate(AMC) 1000, where an Affinity Moiety (AM) 1100 is bound to a medicantmoiety (MM) 1300 via a Linker Unit (LU) 1200, according to variousembodiments of the invention;

FIG. 2A, FIG. 2C, FIG. 2F, FIG. 2H, FIG. 2I, FIG. 2L and FIG. 2M showthe structures of irofulvene medicant moieties, according to variousembodiments of the invention; FIG. 2B, FIG. 2D, FIG. 2E, FIG. 2G, FIG.2J, FIG. 2K, FIG. 2N and FIG. 2O show the structures of illudin medicantmoieties, according to various embodiments of the invention; FIG. 2Pshows the structure of azlactone acyfulvene medicant moiety (where R₂=H,CH₃, CH₂OH), according to an embodiment of the invention; FIG. 2Q showsthe structure of azlactone secondary hydroxyl illudin1 linkage medicantmoiety (where R₂=H, CH₃, CH₂OH), according to an embodiment of theinvention; FIG. 2R shows the structure of azlactone primary hydroxyllinkage illudin2 medicant moiety (where R₂=H, CH₃, CH₂OH), according toan embodiment of the invention; FIG. 2S and FIG. 2T show the structuresof the maleimide acylfulvene and maleimide illudin medicant moieties,respectively; FIG. 2U shows the structure of the maleic acylfulvenemedicant moiety, according to an embodiment of the invention; FIG. 2Vshows the structure of the maleic illudin medicant moiety, according toan embodiment of the invention;

FIG. 3A shows a schematic descriptions of an AMC 1001 where an Antibody(Ab) 1110 bound to a LU 1200 is bound to a medicant moiety (MM) 1300,according to various embodiments of the invention; FIG. 3B shows aschematic descriptions of an AMC 1002 where a Growth factor (GO 1120bound to a LU 1200 is bound to a medicant moiety (MM) 1300, according tovarious embodiments of the invention;

FIG. 4 shows the selective toxicity of an acylfulvene analog, accordingto various embodiments of the invention;

FIG. 5 shows the unique deoxynucleic acid (DNA) damage profile of anacylfulvene analog, according to an embodiment of the invention;

FIG. 6 shows the tumor regression of an acylfulvene analog, according toan embodiment of the invention;

FIG. 7 shows the multidrug resistance studies of an acylfulvene analog,according to an embodiment of the invention;

FIG. 8A shows the structure of the analog 211 attached via the aminogroup using the sulfosuccinimidyl6-(alpha-methyl-alpha-(2-[pyridyldithio)-toluamido)hexanoate (SMPT)linking reagent according to an embodiment of the invention; FIG. 8Bshows the structure of the analog 211 attached via the amino group usingthe sulfosuccimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate(SMCC) linking reagent according to an embodiment of the invention; FIG.8C shows the structure of the analog 211 attached via the amino groupusing the sulfosuccimidyl(4-iodo-acetyl)aminobenzoate (SIAB) linkingreagent according to an embodiment of the invention;

FIG. 9A shows the structure of the analog 038 attached via the carboxylgroup using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDC) linking reagent according to an embodiment of the invention; FIG.9B shows the structure of the analog 038 attached via the carboxyl groupusing the 1-cyclohexyl-3-2(2-morpholinoethyl)carbodiimide (CMC) linkingreagent according to an embodiment of the invention; FIG. 9C shows thestructure of the analog 038 attached via the carboxyl group using theN,N′-dicyclohexylcarbodiimide (DCC) linking reagent according to anembodiment of the invention;

FIG. 10A shows the structure of the analog 038 attached via the carboxylgroup using DCC or N,N′-diisopropylcarbodiimide (DIC) linking reagentsin the presence of glycine according to various embodiments of theinvention; FIG. 10B shows the structure of the analog 038 attached viathe carboxyl group using DCC or DIC linking reagents in the presence ofalanine according to various embodiments of the invention; FIG. 10Cshows the structure of the analog 106 attached via the carboxyl groupusing DCC or DIC linking reagents in the presence of valine according tovarious embodiments of the invention;

FIG. 11A shows the structure of the analog 124 attached via the carbonylgroup using the 2-acetamido-4-mercaptobutyric acid hydrazide (AMBH)linking reagent according to an embodiment of the invention; FIG. 11Bshows the structure of the analog 124 attached via the carbonyl groupusing the p-azidobenzoyl hydrazide (ABH) linking reagent according to anembodiment of the invention; FIG. 11C shows the structure of the analog201 attached via the 4-(N-maleimidomethyl) cyclohexanee-1-1carboxyl-hydrazide (M₂C₂H) linking reagent according to an embodiment ofthe invention;

FIG. 12A shows the structure of the analog 010 attached via the aldehydegroup using the 3-(2-pyridyldithio) propionyl hydrazide (PDPH) linkingreagent according to an embodiment of the invention; FIG. 12B shows thestructure of the analog 010 attached via the aldehyde group using theABH linking reagent according to an embodiment of the invention; FIG.12C shows the structure of the analog 011 attached via4-(4-N-maleimidophenyl)-butyric acid hydrazide (MPBH) linking reagentaccording to an embodiment of the invention;

FIG. 13A shows the structure of the analog 009 attached via the alcoholgroup using the N,N′-carbonyldiimidazole (CDI) linking reagent accordingto an embodiment of the invention; FIG. 13B shows the structure of theanalog 009 attached via the alcohol group using theN-hydroxysuccinimidyl chloroformate (HSC) linking reagent according toan embodiment of the invention; FIG. 13C shows the structure of themedicant moiety Illudin M (FIG. 16A) attached via theN,N′-disuccinimidyl carbonate (DSC) linking reagent according to anembodiment of the invention;

FIG. 14A shows the structure of the analog 051 attached via thesulfhydryl group using SMCC linking reagent according to an embodimentof the invention; FIG. 14B shows the structure of the analog 051attached via the sulfhydryl group using MPBH linking reagent accordingto an embodiment of the invention; FIG. 14C shows the structure of theanalog 051 attached via the sulfhydryl group using PDPH linking reagentaccording to an embodiment of the invention;

FIG. 15A shows the structures of 4-fluorosulfonyl benzoyl; FIG. 15Bshows the structure of 4-fluorosulfonyl benzoyl, 3-fluorosulfonylbenzoyl; FIG. 15C show the structures of 2-fluorosulfonyl benzoyl whereR₁, R₂, R₃, and R₄ independently denote H, F, Cl, Br and I;

FIG. 16A shows the structure of Illudin M; FIG. 16B shows amono-substituted product formed by reacting Illudin M on the secondaryhydroxyl to form 4-FSB linking reagent according to an embodiment of theinvention; FIG. 16C shows a mono-substituted product formed by reactingIlludin M on the secondary hydroxyl to form 2-FSB linking reagentaccording to an embodiment of the invention; FIG. 16D a mono-substitutedproduct formed by reacting Illudin M on the secondary hydroxyl to form3-FSB linking reagent according to an embodiment of the invention;

FIG. 17A shows the structure of the Illudin S FSB mono-substituted onthe primary hydroxy reagent according to an embodiment of the invention;FIG. 17B shows the structure of the Illudin S FSB mono-substituted onthe secondary hydroxy reagent according to an embodiment of theinvention; FIG. 17C shows the structure of the Illudin S FSBdi-substituted reagent according to an embodiment of the invention;

FIG. 18 shows the response of an ADC made by combining analog 316 withthe T101 antibody according to an embodiment of the invention, where thepercent of control is plotted versus the concentration of Namalva, anegative control (i.e., a cell line not expressing T101) where theIC₅₀ >1000 ng/mL and CEM, a positive control (i.e., a cell lineexpressing T101) where the IC₅₀<5 ng/mL, after a four (4) hour exposureand then eighteen (18) hour recovery, where the toxin to antibody ratiois 5:1 (as determined using a radiolabelled toxin) and where theconcentration is in Illudin equivalents (ng of Illudin attached toantibody per mL of cell culture media), which demonstrates the abilityof T101-316 ADC to kill cells expressing T101 antigen on their surfacebut not cells that fail to express the T101 antigen;

FIG. 19A shows the activity (percent survival of the Rituxin antibodyalone is proportional to CD20 expression on B cells (where MV522 cellsare CD20 negative 1910, 8392 cells express low numbers of CD20 1920,Raji express medium numbers of CD20 1930 and Ramos express high numbersof CD20 molecules) 1940, and where B cells are relatively resistant toIlludins and irofulvens (48 hr IC₅₀ >7,000 nM);

FIG. 19B shows the activity of the Rituxin antibody alone 1950 comparedwith an ADC of Rituxin with analog 218 on Ramos cells 1955;

FIG. 19C shows the activity of the Rituxin antibody alone 1960 comparedwith an ADC of Rituxin with analog 218 on Raji cells 1965;

FIG. 19D shows the activity of the Rituxin antibody alone 1970 comparedwith an ADC of Rituxin with analog 218 on 8392 cells 1975; FIG. 19Eshows the activity of the Rituxin antibody alone 1980 compared with anADC of Rituxin with analog 218 on MV522 cells (where MV522 cells grew inthe presence of the Rituxan-analog 218 ADC but at a slower rate than inthe absence of analog 218 ADC) 1985).

FIG. 20A shows an illudin analog according to various embodiments of theinvention; FIG. 20B shows a syn-illudin analog according to variousembodiments of the invention;

FIG. 20C and FIG. 20D show acylfulvene analogs according to variousembodiments of the invention;

FIG. 21A shows analog 20 linked to DSP according to an embodiment of theinvention;

FIG. 21 shows analog 20 linked to DTME according to an embodiment of theinvention; and

FIG. 21 shows analog 20 linked to SMPT according to an embodiment of theinvention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “receptor for a biologically activepolypeptide” means a receptor which can bind a biologically activepeptide conjugate.

As used herein, the term “cell population” is used to describe a set orsubset of cells expressing a receptor.

As used herein, the terms “analog”, “medicant” and “medicant moiety” areused interchangeably and comprise synthetic and naturally occurringdrugs, toxins, nutraceuticals and other cytoactive, anti-inflammatoryand bioactive molecules including Doxorubicin (Immunomedics),auristatins E (Seattle Genetics), auristatins F (Celdex), monomethylauristatin E (MMAE) (Amgen), monomethyl auristatin F (MMAF) (Astelles),maytanasines (Immunogen), DM1 (Biotest), DM4 (Amgen), calicheamicin(CellTech), irinotecan, folate, SN38 (Immunomedics),Pyrrolobenzodiazepines (Seattle Genetics), MGBA a duocarmycin derivative(Medarex), thalidomides, taxanes, penicillins, Trastuzumab emtansine(Genentech for Breast cancer uses maytanasine derive DM-1). A medicantincludes analogs 192, 197, 272, 273, 274, 290, 291, 292, 293 (i.e.,acylfulvenes linked to thalidomide, cephalosporin or colchicinesderivatives). Some of the above analogs are stand alone drugs, but canbe used as a medicant moiety in an affinity drug conjugate according tovarious embodiments of the invention.

As used herein, the phrase “peptide receptor” includes peptide hormonereceptors, protein hormone receptors, chemotactic receptors andchemokine receptors.

As used herein, the term “receptor” includes growth factor receptors,peptide hormone receptors, peptide receptors, steroid hormone receptors,steroid receptors and lipid receptors.

As used herein, phrase “affinity medicant conjugate” is an AffinityMoiety covalently bound to a medicant moiety, and includes antibodymedicant conjugates, where the antibody is directed to a specificreceptor. As used herein the phrase ‘Affinity Moiety’ includesantibodies, antibody fragments, peptides, proteins, growth factors,steroids, and lipids, where the antibodies, antibody fragments,peptides, proteins, growth factors, steroids, folate or lipids have anaffinity for a specific receptor, receptors, is processed by an enzymeto produce a ligand that has an affinity for a specific receptor orotherwise directs the Affinity Moiety to a specific subset of cells. A‘medicant moiety’ includes a group bound to an Affinity Moiety, whichwhen released acts as a medicant.

As used herein, the term “Affinity Moiety” (AM) is used to describe achemical group or molecule that can bind a receptor or proteins. An AMis understood to have a minimum binding affinity greater thanapproximately 1×10⁻³ M affinity. As used herein, the term AM includes“ligands”, “ligand moieties”, “affinity unit” and an AM modified toinclude a linker. As used herein, the phrase “an affinity moietydirected to a peptide receptor” is used to describe a molecule or aportion of a molecule which has a binding affinity to the peptidereceptor greater than approximately 1×10⁻¹° M. In this rangeapproximately means 1×10⁻⁹ M to 1×10⁻¹¹ M. In an embodiment of theinvention, an AM directed to a peptide receptor has a binding affinityto the peptide receptor greater than approximately 1×10⁻¹² M. In thisrange approximately means 1×10⁻¹¹ M to 1×10⁻¹³ M.

As used herein, the term “linker” is used to describe one or morecovalently bonded groups of atoms that are covalently bonded to amedicant moiety and an AM. For example a linker can be covalently boundto both an acylfulvene moiety and to an antibody or other ligand moietywith an affinity for a receptor.

As used herein, the term “non releasable linker” is used to describe alinker covalently bound to an AM and a medicant moiety in which the AMand the medicant moiety remain covalently bound to the linker afterinternalization and exposure to both reducing and acidic environments ofvesicles within the cell. As used herein, the term “membranepermeability” is used to describe a compound comprising a linkercovalently bound to an AM and an acylfulvene moiety, where the compoundcan diffuse across membranes within the cell.

As used herein, the term “transmembrane receptor” means a protein thatspans the plasma membrane of a cell with the extracellular domain of theprotein having the ability to bind an AM and the intracellular domainhaving an activity such as activation of G protein signaling which isinduced upon the AM binding.

As used herein, the term “seven transmembrane receptor” is atransmembrane receptor including a transmembrane domain where theprotein spans the cell membrane in seven (7) regions.

As used herein, the term “G-protein coupled receptor” means a seventransmembrane domain receptor which transduces a biological signal viaG-protein coupling.

As used herein, the term “conjugated” or “conjugate” means a chemicalcompound that is formed by joining two or more compounds with one ormore chemical bonds or linkers. In an embodiment of the invention, anantibody and a medicant form a conjugate.

As used herein, the term “antibody” herein is used in the broadest senseand specifically covers intact antibodies, monoclonal antibodies,polyclonal antibodies, mono-specific antibodies, multi-specificantibodies (e.g., bi-specific antibodies), and antibody fragments thatexhibit the desired biological activity, including those antibodiesdirected against Alk, Alk fusion proteins, CD 2 (SEQ. ID. 001),CD3epsilon (SEQ. ID. 002), CD5 (SEQ. ID. 003), CD7 (SEQ. ID. 004), CD19(SEQ. ID. 005), CD20 (SEQ. ID. 006), CD22 (SEQ. ID. 007), CD25 (SEQ. ID.008), CD30 (SEQ. ID. 009), CD33 (SEQ. ID. 010), CD37 (SEQ. ID. 011),CD44 (SEQ. ID. 012), CD44v6 (SEQ. ID. 013), CD56 (SEQ. ID. 014), CD70(SEQ. ID. 015), CD74 (SEQ. ID. 016), CD79 (SEQ. ID. 017), CD79b (SEQ.ID. 018), CD 80 (SEQ. ID. 019), CD 86 (SEQ. ID. 020), CD138 (syndecan 1)(SEQ. ID. 021), CAIX (SEQ. ID. 022), Integrin alphaVbeta 3 (SEQ. ID.023), EphA2 (SEQ. ID. 024), Crypto1 (SEQ. ID. 025), CanAg (SEQ. ID.026), ENPP3 (SEQ. ID. 027), Nectin-4 (SEQ. ID. 028), Mesothelin (SEQ.ID. 029), Lewis Y (SEQ. ID. 030), EGFRvIII (SEQ. ID. 031), SLC44A4 (SEQ.ID. 032), EBTR (endothelin) (SEQ. ID. 033), erbB2/neu/HER2 (SEQ. ID.034), Transferrin receptor (SEQ. ID. 035), 55 kDa breast cancer antigen,72 kDa TAA, GPNMB (osteoactivin) (SEQ. ID. 038), CA-IX (SEQ. ID. 039),CEA (CD66e) (SEQ. ID. 040), CEACAMS (SEQ. ID. 041), PSMA (SEQ. ID. 042),CA125 (MUC16) (SEQ. ID. 043), Mucl (CA6) (SEQ. ID. 044), Melanomaglycoprotein NMB (SEQ. ID. 045), IL-2R (SEQ. ID. 166 and 046), IL13R(SEQ. ID. 047), TACSTD2 (TROP2 or EGP1) (SEQ. ID. 048), Folate receptor1 (SEQ. ID. 049), Mucin 16 (SEQ. ID. 050), Endothelin receptor ETB (SEQ.ID. 051), STEAP1 (SEQ. ID. 052), SLC44A4 (AGS-5) (SEQ. ID. 053), AGS-16(SEQ. ID. 054), and Guanylyl cyclase C (SEQ. ID. 055). An intactantibody has primarily two regions: a variable region and a constantregion. The variable region binds to and interacts with a targetantigen. The variable region includes a complementary determining region(CDR) that recognizes and binds to a specific binding site on aparticular antigen. The constant region may be recognized by andinteract with the immune system. An antibody can be of any type or class(e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2). The antibody can be derived from any suitablespecies. In some embodiments, the antibody is of human or murine origin.An antibody can be, for example, human, humanized or chimeric.

As used herein, the terms “specifically binds” and “specific binding”refer to antibody binding to a predetermined antigen. Typically, theantibody binds with an affinity of at least about 1×10⁷ M, and binds tothe predetermined antigen with an affinity that is at least two-foldgreater than its affinity for binding to a non-specific antigen (e.g.,Bovine Serum Albumin, casein) other than the predetermined antigen or aclosely-related antigen.

As used herein, “isolated” means separated from other components of (a)a natural source, such as a plant or animal cell or cell culture, or (b)a synthetic organic chemical reaction mixture. As used herein,“purified” means that when isolated, the isolate contains at least 95%,and in another aspect at least 98%, of a compound (e.g., a conjugate) byweight of the isolate.

As used herein, the term “therapeutically effective amount” refers to anamount of a medicant effective to treat a disease or disorder in amammal. In the case of cancer, the therapeutically effective amount ofthe medicant may reduce the number of cancer cells; reduce the tumorsize; inhibit (i.e., slow to some extent and preferably stop) cancercell infiltration into peripheral organs; inhibit (i.e., slow to someextent and preferably stop) tumor metastasis; inhibit, to some extent,tumor growth; and/or relieve to some extent one or more of the symptomsassociated with the cancer. To the extent the medicant may inhibit thegrowth of and/or kill existing cancer cells, it may be cytostatic and/orcytotoxic. For cancer therapy, efficacy can, for example, be measured byassessing the time to disease progression (TTP) and/or determining theresponse rate (RR).

As used herein, the term “substantial amount” refers to a majority, i.e.greater than approximately fifty percent (50%) of a population, of amixture or a sample. In this range approximately means plus or minus tenpercent (10%).

As used herein, the term “intracellular metabolite” refers to a compoundresulting from a metabolic process or reaction inside a cell on anAffinity Medicant Linker conjugate (e.g., an Antibody Drug Conjugate(AMC)). The metabolic process or reaction may be an enzymatic processsuch as proteolytic cleavage of a peptide linker of the AMC.Intracellular metabolites include, but are not limited to, antibodiesand free medicant which have undergone intracellular cleavage afterentry, diffusion, uptake or transport into a cell.

As used herein, the terms “intracellularly cleaved” and “intracellularcleavage” refer to a metabolic process or reaction inside a cell on anAffinity Medicant Linker conjugate (e.g., an Antibody Medicant conjugate(AMC) or the like), whereby the covalent attachment, e.g., the linker,between the Medicant moiety (M) and the Affinity unit (e.g., an antibody(Ab)) is broken, resulting in the free Medicant, or other metabolite ofthe conjugate dissociated from the antibody inside the cell. The cleavedmoieties of the Affinity Medicant Linker conjugate are thusintracellular metabolites.

As used herein, the term “bioavailability” refers to the systemicavailability (i.e., blood/plasma levels) of a given amount of a medicantadministered to a patient. Bioavailability is an absolute term thatindicates measurement of both the time (rate) and total amount (extent)of medicant that reaches the general circulation from an administereddosage form.

As used herein, the term “cytotoxic activity” refers to a cell-killing,a cytostatic or an anti-proliferative effect of an Affinity MedicantLinker conjugate or an intracellular metabolite of an Affinity MedicantLinker conjugate. Cytotoxic activity may be expressed as the IC₅₀ value,which is the concentration (molar or mass) per unit volume at which halfthe cells survive.

As used herein, the term “cytotoxic agent” as used herein refers to asubstance that inhibits or inhibits the function of cells and/or causesdestruction of cells. The term is intended to include radioactiveisotopes (e.g., ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P,⁶⁰C, and radioactive isotopes of Lu), chemotherapeutic agents, andtoxins such as small molecule toxins or enzymatically active toxins ofbacterial, fungal, plant or animal origin, including synthetic analogsand derivatives thereof. In one aspect, the term does not include aradioactive isotope(s).

As used herein, an example of a “patient” includes, but is not limitedto, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog,cat, bird and fowl. In an exemplary embodiment, the patient is a human.

As used herein, the terms “treat” or “treatment,” unless otherwiseindicated by context, refer to therapeutic treatment and prophylacticmeasures to prevent relapse, wherein the object is to inhibit or slowdown (lessen) an undesired physiological change or disorder, such as thedevelopment or spread of cancer. For purposes of this invention,beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already having the condition or disorder as wellas those prone to have the condition or disorder.

As used herein, in the context of cancer, the term “treating” includesany or all of inhibiting growth of tumor cells, cancer cells, or of atumor; inhibiting replication of tumor cells or cancer cells, lesseningof overall tumor burden or decreasing the number of cancerous cells, andameliorating one or more symptoms associated with the disease.

As used herein, the term “chiral” refers to molecules which have theproperty of non-superimposability of the mirror image partner, while theterm “achiral” refers to molecules which are superimposable on theirmirror image partner.

As used herein, the term “stereoisomers” refers to compounds which haveidentical chemical constitution, but differ with regard to thearrangement of the atoms or groups in space.

As used herein, “diastereomer” refers to a stereoisomer with two or morecenters of chirality and whose molecules are not mirror images of oneanother. Diastereomers have different physical properties, e.g., meltingpoints, boiling points, spectral properties, and reactivities. Mixturesof diastereomers may separate under high resolution analyticalprocedures such as electrophoresis and chromatography.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedchain, or combination thereof, which may be fully saturated, mono- orpolyunsaturated and can include di- and multivalent radicals, having thenumber of carbon atoms designated (i.e., C₁-C₁₀ means one to tencarbons). Examples of saturated hydrocarbon radicals include, but arenot limited to, groups such as methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs andisomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and thelike. An unsaturated alkyl group is one having one or more double bondsor triple bonds. Examples of unsaturated alkyl groups include, but arenot limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy isan alkyl attached to the remainder of the molecule via an oxygen linker(—O—).

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred in the presentinvention. A “lower alkyl” or “lower alkylene” is a shorter chain alkylor alkylene group, generally having eight or fewer carbon atoms.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcombinations thereof, consisting of at least one carbon atom and atleast one heteroatom selected from the group consisting of O, N, P, Si,and S, and wherein the nitrogen and sulfur atoms may optionally beoxidized, and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) O, N, P, S, and Si may be placed at any interior positionof the heteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. Examples include, but are notlimited to: —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH—₂—CH₃, and—CN. Up to two heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—. As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where“heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R″ or the like, it will be understoodthat the terms heteroalkyl and —NR′R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl,” respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl,and the like. Examples of heterocycloalkyl include, but are not limitedto, 1-(1,2,5,6-tetrahydropyridyl), 1 piperidinyl, 2-piperidinyl, 3piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,and 3-bromopropyl.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. The term “heteroaryl” refers to aryl groups (or rings)that contain from one to four heteroatoms selected from N, O, and S,wherein the nitrogen and sulfur atoms are optionally oxidized, and thenitrogen atom(s) are optionally quaternized. Thus, the term “heteroaryl”includes fused ring heteroaryl groups (i.e., multiple rings fusedtogether wherein at least one of the fused rings is a heteroaromaticring). A 5,6-fused ring heteroarylene refers to two rings fusedtogether, wherein one ring has 5 members and the other ring has 6members, and wherein at least one ring is a heteroaryl ring. Likewise, a6,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 6 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylenerefers to two rings fused together, wherein one ring has 6 members andthe other ring has 5 members, and wherein at least one ring is aheteroaryl ring. A heteroaryl group can be attached to the remainder ofthe molecule through a carbon or heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. An “arylene” and a“heteroarylene,” alone or as part of another substituent, mean adivalent radical derived from an aryl and heteroaryl, respectively.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl, and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

The term “alkylsulfoxide” as used herein, means a moiety having theformula R—S(O)—R′, where R and R′ are alkyl groups as defined above. Rand R′ may have a specified number of carbons (e.g., “C₁-C₄alkylsulfoxide”).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl,” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN, and—NO₂ in a number ranging from zero to (2m′+1), where m is the totalnumber of carbon atoms in such radical. R′, R″, R′″, and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g.,aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl,alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound ofthe invention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″, and R″″ groupwhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ includes, but is not limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″ C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂,fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in a number ranging fromzero to the total number of open valences on the aromatic ring system;and where R′, R″, R′″, and R″″ are preferably independently selectedfrom hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″,and R″″ groups when more than one of these groups is present.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′—(C″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties: (A) —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen,unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,unsubstituted heteroaryl, and (B) alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, and heteroaryl, substituted with at least onesubstituent selected from: (i) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂,halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,unsubstituted heteroaryl, and (ii) alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, and heteroaryl, substituted with at least onesubstituent selected from: (a) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂,halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,unsubstituted heteroaryl, and (b) alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl, substituted with at least onesubstituent selected from: oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂,halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, andunsubstituted heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₄-C₈cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 4 to 8 membered heterocycloalkyl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds that are prepared with relatively nontoxic acidsor bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and thelike. Also included are salts of amino acids such as arginate, and saltsof organic acids like glucuronic or galacturonic acids. Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

Thus, the compounds of the present invention may exist as salts, such aswith pharmaceutically acceptable acids. The present invention includessuch salts. Examples of such salts include hydrochlorides,hydrobromides, sulfates, methanesulfonates, nitrates, maleates,acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates,(−)-tartrates, or mixtures thereof including racemic mixtures),succinates, benzoates, and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in theart.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

In addition to salt forms, the present invention provides compounds in aprodrug form. Prodrugs of the compounds described herein are thosecompounds that readily undergo chemical changes under physiologicalconditions to provide the compounds of the present invention.Additionally, prodrugs can be converted to the compounds of the presentinvention by chemical or biochemical methods in an ex vivo environment.For example, prodrugs can be slowly converted to the compounds of thepresent invention when placed in a transdermal patch reservoir with asuitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,tautomers, geometric isomers, and individual isomers are encompassedwithin the scope of the present invention. The compounds of the presentinvention do not include those that are known in the art to be toounstable to synthesize and/or isolate.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I), carbon-13 (¹³C), or carbon-14 (¹⁴C). Allisotopic variations of the compounds of the present invention, whetherradioactive or not, are encompassed within the scope of the presentinvention.

Many organic compounds exist in optically active forms, i.e., they havethe ability to rotate the plane of plane-polarized light. In describingan optically active compound, the prefixes D and L, or R and S, are usedto denote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and 1 or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or 1 meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesestereoisomers are identical except that they are mirror images of oneanother. A specific stereoisomer may also be referred to as anenantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

As used herein, an amino acid “derivative” includes an amino acid havingsubstitutions or modifications by covalent attachment of a parent aminoacid, such as, e.g., by alkylation, glycosylation, acetylation,phosphorylation, and the like. Further included within the definition of“derivative” is, for example, one or more analogs₁ of an amino acid withsubstituted linkages, as well as other modifications known in the art.

As used herein, a “natural amino acid” refers to arginine, glutamine,phenylalanine, tyrosine, tryptophan, lysine, glycine, alanine,histidine, serine, proline, glutamic acid, aspartic acid, threonine,cysteine, methionine, leucine, asparagine, isoleucine, and valine,unless otherwise indicated by context.

As used herein, a “protecting group” refers to a moiety that whenattached to a reactive group in a molecule masks, reduces or preventsthat reactivity. Representative hydroxy protecting groups include acylgroups, benzyl and trityl ethers, tetrahydropyranyl ethers,trialkylsilyl ethers and allyl ethers. Representative amino protectinggroups include formyl, acetyl, trifluoroacetyl, benzyl,benzyloxycarbonyl (CBZ), tert-butoxycarbonyl (Boc), trimethyl silyl(TMS), 2-trimethylsilyl-ethanesulfonyl (SES), trityl and substitutedtrityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC),nitro-veratryloxycarbonyl (NVOC), and the like. Examples of a “hydroxylprotecting group” include, but are not limited to, methoxymethyl ether,2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether,p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether,triisopropyl silyl ether, t-butyldimethyl silyl ether, triphenylmethylsilyl ether, acetate ester, substituted acetate esters, pivaloate,benzoate, methanesulfonate and p-toluenesulfonate.

Abbreviations used include: DMAP=4-dimethylaminopyridine;DCC=N,N′-dicycyclohexylcarbodiimide;ODHBt=3,4,-dihydroxy-4-oxo-1,2,3-benzo-triazine-3-yl ester;NMM=N-methylmorpholin; EDC=1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride; DIAD=diisopropyl azodicarboxylate; DEAD=diethylazodicarboxylate; and DIPC=N,N′-diisopropylcarbodiimide.

As used herein, a “leaving group” refers to a functional group that canbe substituted by another functional group. Such leaving groups are wellknown in the art, and examples include, but are not limited to, a halide(e.g., chloride, bromide, and iodide), methanesulfonyl (mesyl),p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), andtrifluoromethylsulfonate.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound(e.g., a Medicant Linker compound, or an Affinity Medicant Linkerconjugate). The compound typically contains at least one amino group,and accordingly acid addition salts can be formed with this amino group.Exemplary salts include, but are not limited to, sulfate, citrate,acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acid phosphate, isonicotinate, lactate, salicylate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucuronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and palmoate(i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Apharmaceutically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counter ion.The counter ion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a pharmaceuticallyacceptable salt may have more than one charged atom in its structure.Instances where multiple charged atoms are part of the pharmaceuticallyacceptable salt can have multiple counter ions. Hence, apharmaceutically acceptable salt can have one or more charged atomsand/or one or more counter ion.

As used herein, a “pharmaceutically acceptable solvate” or “solvate”refer to an association of one or more solvent molecules and a compoundof the invention, e.g., an Affinity Medicant Linker conjugate or aMedicant Linker compound. Examples of solvents that formpharmaceutically acceptable solvates include, but are not limited to,water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid,and ethanolamine.

The following abbreviations are used herein and have the indicateddefinitions: Boc is N-(t-butoxycarbonyl), cit is citrulline, dap isdolaproine, DCM is dichloromethane, DIEA is N,N-diisopropylethylamine,dil is dolaisoleuine, DMF is N,N-dimethylformamide, DMSO isdimethylsulfoxide, doe is dolaphenine, dov is N,N-dimethylvaline, DTNBis 5,5′-dithiobis(2-nitrobenzoic acid), DTPA isdiethylenetriaminepentaacetic acid, DTT is dithiothreitol, Fmoc isN-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU isO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, HBTU is2-[1H-benzotriazole-1-yl]-1,1,3,3-tetramethylaminiumhexafluorophosphate; HOBt is 1-hydroxybenzotriazole, HPLC is highpressure liquid chromatography, ile is isoleucine, lys is lysine, MeOHis methanol, MeVal is N-methyl-valine, PAB is p-aminobenzyl, PBS isphosphate-buffered saline (pH 7.4), Ph is phenyl, phe isL-phenylalanine, PyBrop is bromo tris-pyrrolidino phosphoniumhexafluorophosphate, TFA is trifluoroacetic acid, UV is ultraviolet, andval is valine.

The following LU abbreviations are used herein and have the indicateddefinitions: Val Cit or vc is a valine-citrulline dipeptide site inprotease cleavable linker; PABC is p-aminobenzylcarbamoyl; (Me)vc isN-methyl-valine citrulline, where the linker peptide bond has beenmodified to prevent its cleavage by cathepsin B; and MC(PEG)₆-OH ismaleimidocaproyl-polyethylene glycol.

As used herein, a “pegylated compound” refers to a compound conjugatedwith two or more polyethylene glycol moieties or two or morepolypropylene glycol moieties or a combination thereof.

As used herein, a “pro-peptide” includes pro-peptide, pre-peptide,pro-protein and pre-protein amino acid sequences including those aminoacid sequences cleaved by enzymes disclosed in Table IX.

As used herein, “Illudin1” or “illudin-1” means those analogs disclosedin Table XI. As used herein “Illudin2” or “illudin2” means those analogsdisclosed in Table XI and Table XII. As used herein, “acylfulvene” means“illudin2” and any analog derived therefrom.

Malignant neoplasia is the second most common cause of death in theUnited States behind cardiovascular disease. Chemotherapy has exerted apredominant role in increasing life spans for patients with a variety oftumors including Burkitt's lymphoma, acute lymphocytic leukemia andHodgkin's disease. Further, new cancer chemotherapeutic agents andmethods of care combined with early detection and treatment haveresulted in decreases in the overall incidence of cancer and decreasesin the death rates from all cancers combined. Responsive tumorsrepresent only a small fraction of the various types of cancer. Further,agents such as cyclophosphamide, adriamycin, 5-fluorouracil andhexamethylmelamine, which are highly active against clinical solidtumors, are limited. Thus, patients with many types of malignanciesremain at significant risk for relapse and mortality. After relapse,some patients can be re-induced into remission with their initialtreatment regimen. However, higher doses of the initial chemotherapeuticagent or the use of additional agents are frequently required,indicating the development of at least partial medicant resistance.Evidence indicates medicant resistance can develop simultaneously toseveral agents, including medicant resistance to treatments to which thepatient was not exposed. The development of multiple-medicant resistanttumors may be a function of tumor mass and constitutes a major cause oftreatment failure. To overcome this medicant resistance, high-dosechemotherapy with or without radiation and allogenic or autologous bonemarrow transplantation can be employed. The high-dose chemotherapy mayemploy the original medicant(s) or be altered to include additionalagents. As a result, there remain many cancer patients for whom no orminimally effective therapy exists. Accordingly, there is a need for thedevelopment of novel chemotherapeutics with greater efficacy or safety,either as monotherapy or in combination with other chemotherapeuticagents, and such agents with the potential to overcome medicantresistance in cancer cells.

Illudins are toxic natural products produced by mushrooms of the genusOmphalotus (FIG. 20A). Syn-Illudins are semi-synthetic derivatives ofIlludins. Acylfulvenes are also semi-synthetic derivatives of Illudins.Syn-Illudins and Acylfulvenes have each been chemically modified atselect sites to allow their use as medicants. The modifications in theSyn-Illudins do not alter any of the cyclic rings (cyclopropane,cyclopentane, cyclohexane) of the basic Illudin chemical structure (FIG.20B). The modifications of Acylfulvenes differ from Syn-Illudins in thatan additional double bond (an unsaturated bond) has been created in the5 membered (cyclopentane) ring (FIG. 20C, FIG. 20D).

Illudins function as alkylating agents that damage DNA and thereby blocktranscription. The blockage can be repaired through nucleotide excision.The toxicity of the illudins has prevented any applications in humantumor therapy. Acylfulvenes have been developed which exhibit promisingantitumor activity with a better safety profile, as described in U.S.Pat. Nos. 5,439,936; 5,523,490 and 6,380,403 which are each hereinexpressly incorporated by reference in their entireties. Irofulven or6-hydroxymethylacylfulvene (see FIG. 6) is an analog of illudin S whichhas demonstrated clinical activity with an acceptable safety profile inhormone-refractory prostate cancer. Most relevant to clinicalapplications, irofulven activity is independent of common resistancemechanisms such as the multi-medicant resistance phenotype,anti-apoptotic B-cell lymphoma 2 (Bcl-2) (SEQ. ID. 056) over expression,as well as tumor protein 53 (p53) (SEQ. ID. 057) and cyclin dependentkinase inhibitor 1 (p21/WAF1) (SEQ. ID. 058) mutations (see FIG. 7 andTable XIV).

Growth factors, including peptides and proteins are critical mediatorsof a wide range of cell-cell communication. They are importantendocrine, paracrine and autocrine messengers. Growth factors functionas neurotransmitters and neuromodulators, regulate chemotaxis, immunefunction, development, cell growth, and can influence tumor cells. Thereceptors that recognize growth factors are highly selective and definespecific cell populations. As a result, growth factor receptors are alarge and important class of medicant (including drug) targets. Inaddition to physiologic noncancerous cell populations, these receptorscan also be expressed in various cancer cell populations.

A polypeptide is a long, continuous, and unbranched chain of aminoacids. A glycol-peptide is a peptide that contains one or morecarbohydrate moieties covalently attached to the side chains of specificamino acids. A pro-peptide, is an inactive peptide that can be turnedinto an active form through a post translational modification thatenzymatically cleaves the pro-peptide. Examples include pro-insulin(SEQ. ID. 059) and pro-opiomelanocortin (SEQ. ID. 060). Enzymaticallycleaving the pro-peptide, allows for the peptide to be available onshort notice and/or in large quantities. Some pro-peptides are secretedfrom the cell. Many of these are synthesized with an N-terminal signalpeptide that targets the pro-peptide for secretion.

Cytokines are small proteins (approximately 5 to 20 kDa) that affect thebehavior of other cells, and sometimes the releasing cell itself and arethereby important in cell signaling (see Table XIV). Many specificcytokines can be released by a variety of different kinds of cells,e.g., macrophages, B lymphocytes, T lymphocytes, mast cells, endothelialcells, fibroblasts, and various stromal cells. Cytokines act throughspecific receptors, and are important in the humoral and cell-basedimmune responses. Cytokines also regulate the maturation, growth, andresponsiveness of specific cell populations. Cytokines circulate in muchhigher concentrations than hormones and in contrast with hormones aremade by a variety of different kinds of cells. Cytokines are importantin host responses to infection, immune responses, inflammation, trauma,sepsis, cancer, and reproduction. As a result, cytokine receptors areupregulated in many forms of cancers.

A steroid is an organic compound that contains four cycloalkane ringsjoined to each other. Examples of steroids include the dietary lipidcholesterol and the sex hormones estradiol and testosterone. The core ofa steroid molecule is composed of seventeen carbon atoms bonded togetherthat take the form of four fused rings:three six-carbon atom rings andone five-carbon atom ring. A variety of functional groups can beattached to the four-ring core. Steroids can also vary depending on theoxidation state of the rings. A steroid hormone is a steroid that actsas a hormone. Steroid hormones can be grouped into five groups(glucocorticoids, mineralocorticoids, androgens, estrogens, andprogesterones) based on the receptors to which they bind. Steroidhormones, particularly androgens, are essential not only for growth anddevelopment but also in the progression of many forms of cancer. As aresult, steroid hormone receptors are upregulated in many forms ofcancers.

The retinoic acid receptor (RAR) is a nuclear receptor which can alsoact as a transcription factor. The RAR can be activated by eitherall-trans retinoic acid or 9-cis retinoic acid. There are three RARisoforms (alpha (SEQ. ID. 061), beta (SEQ. ID. 062), and gamma (SEQ. ID.063)), each encoded by separate genes, where splice variants generatestill further diversity in the expressed receptor. The retinoid Xreceptor (RXR) is a nuclear receptor activated by 9-cis retinoic acid.There are also three RXR isoforms (alpha (SEQ. ID. 064), beta (SEQ. ID.065), and gamma (SEQ. ID. 066)), each encoded by separate genes. RXRhetero-dimerizes with subfamily 1 nuclear receptors including RAR. Inthe absence of ligand, the RAR/RXR dimer binds to retinoic acid responseelements complexes with a co-repressor protein. Binding of agonistligands to RAR results in dissociation of the co-repressor andrecruitment of a co-activator protein that, in turn, promotestranscription of the downstream target gene into mRNA and therebyprotein or other RNA signaling mechanisms.

Lipid metabolism is altered in many forms of cancer, includingupregulation of de novo lipid synthesis. Cancer cells can also usealternative enzymes and pathways to facilitate the production of fattyacids. These newly synthesized lipids may support a number of cellularprocesses to promote cancer cell proliferation and survival. Elaidicacid or (E)-octadec-9-enoic acid is the trans isomer of oleic acid andis found in small quantities in caprine milk, bovine milk and somemeats. It increases Cholesteryl Ester Transfer Protein (CETP) (SEQ. ID.067) activity, which in turn raises levels of very low densitylipoprotein and lowers levels of high density lipoprotein (HDL)cholesterol. CETP is found in plasma, where it is involved in thetransfer of cholesteryl ester from HDL to other lipoproteins. Defects inthe CETP gene are a cause of hyperalphalipoproteinemia 1.

An antibody is a protein made up of four peptide chains disulfide linkedtogether to form a “Y”-shape. Antibodies are produced by plasma cellsand are used by the immune system to identify and neutralize foreignantigens such as bacteria and viruses. The antibody recognizes a uniquepart of the antigen using each FAB portion of the protein (i.e., the tipof the “Y” portion of the antibody), allowing a specific high affinitybinding interaction to occur. The binding interaction of differentantibodies can target specific antigen epitopes. An antibody fragmentcontaining one or both FAB portions can also target specific antigenepitopes.

The ability of the Illudin, Syn-Illudin and Acylfulvene analogs toinhibit tumor cell growth is shown in Table XV. The MV522 cell line is alung-derived adenocarcinoma cell line. In various embodiments of theinvention, the MV522 cell line represents a “target” cell line. That isan Illudin, Syn-Illudin or Acylfulvene analog that exhibits toxicityagainst this solid tumor cell line shows a desirable result. The 8392Bcell line represents a hematopoietic (non-solid) cell line. In variousembodiments of the invention, the 8392B cell line is considered a“nontarget” cell line. The two hour toxicity data represents theconcentration of a given analog for which a two hour exposure willinhibit 50% of the DNA synthesis activity in a given cell line. The 48hour exposure data represents the concentration at which a given analogwith a 48 hour exposure will inhibit the growth or viability in a givencell line as defined by the standard Trypan Blue Exclusion assay. As anexample, analog 002 will inhibit the target MV522 cell line at 110 nMwith only a 2 hour exposure but has no inhibitory effect on thenontarget 8392B cell line at 26,000 nM (26 μM). Analog 002 with aprolonged exposure period (e.g. 48 hours) can eventually inhibit thenontarget cell line. In contrast, Analog 201 will inhibit the targetMV522 cell line with only a 2 hour exposure (IC50=360 nM) but hasminimal effect on the 8392B cell nontarget line with even a 48 hourexposure (IC50=26,000 nM) indicating superior anticancer activity as amonotherapeutic agent. In contrast to these two analogs, analog 224displayed minimal toxicity as well as no differential toxicity betweenthe target and nontarget cell line indicating it would have minimalproperties as a monotherapeutic anticancer agent.

As used herein, a “growth factor” or an “anti-angiogenic protein”includes Adrenomedullin (SEQ. ID. 068), Angiopoietin (Ang) (SEQ. ID.069, 106, 111, and 145), Autocrine motility factor (SEQ. ID. 070), Bonemorphogenetic proteins (BMPs) (SEQ. ID. 071), Brain-derived neurotrophicfactor (BDNF) (SEQ. ID. 072), Endostatin (SEQ. ID. 073), Endostar (SEQ.ID. 074), Epidermal growth factor (EGF) (SEQ. ID. 075), Erythropoietin(EPO) (SEQ. ID. 076), Fibroblast growth factor (FGF) (SEQ. ID. 077),Glial cell line-derived neurotrophic factor (GDNF) (SEQ. ID. 078),Granulocyte colony-stimulating factor (G-CSF) (SEQ. ID. 079),Granulocyte macrophage colony-stimulating factor (GM-CSF) (SEQ. ID.080), Growth differentiation factor-9 (GDF9) (SEQ. ID. 081), Hepatocytegrowth factor (HGF) (SEQ. ID. 082), Hepatoma-derived growth factor(HDGF) (SEQ. ID. 083), Insulin-like growth factor (IGF) (SEQ. ID. 084),Migration-stimulating factor (SEQ. ID. 085), Myostatin (GDF-8) (SEQ. ID.086), Nerve growth factor (NGF) (SEQ. ID. 087) and other neurotrophins(SEQ. ID. 144), Platelet-derived growth factor (PDGF A) (SEQ. ID. 088),PDGF B (SEQ. ID 168), PDGF C (SEQ. ID. 036), PDGF D (SEQ. ID. 037),Thrombopoietin (TPO) (SEQ. ID. 089), Transforming growth factoralpha(TGF-α) (SEQ. ID. 090), Transforming growth factor beta(TGF-β)(SEQ. ID. 091), Tumor necrosis factor-alpha(TNF-α) (SEQ. ID. 092),Vascular endothelial growth factor (VEGF) (SEQ. ID. 093), and placentalgrowth factor (P1GF) (SEQ. ID. 094).

As used herein, a “protein toxin” includes ricin A chain (SEQ. ID. 095),ricin B chain (SEQ. ID. 096), diphtheria toxin (SEQ. ID. 097),Pseudomonas aeurginosa exotoxin A (SEQ. ID. 098), r-gelonin (SEQ. ID.099), saporin (SEQ. ID. 100), glycosylated protein toxins, deglcosylatedprotein toxins and protein toxin fragments which includes deglycosylatedricin A, deglycosylated ricin B, Pseudomonas aeurginosa exotoxin A PE40fragment (SEQ. ID. 101) and Pseudomonas aeurginosa exotoxin A PE38fragment (SEQ. ID. 102).

As used herein, a “steroid” includes cholesterol (5-cholesten-3beta-ol),pregnenolone (3beta-hydroxy-5-pregnen-20-one), 17-hydroxyprenenolone(3-beta,17-dihydroxy-5-pregnen-20-one), progesterone(4-pregnene-3,20-dione), 17-hydroxyprogesterone(17-hydroxy-4-pregnene-3,20-dione), androstenedione(4-androstene-3,17-dione), 4-hydroxyandrostenedione(4-hydroxy-4-androstene-3,17-dione), 11-beta-hydroxyandostenedione(11beta-4-androstene-3,17-dione), androstanediol(3-beta,17-beta-Androstanediol), androsterone(3-alpha-hydroxy-5alpha-androstan-17-one), epiandrosterone(3-beta-hydroxy-5alpha-androstan-17-one), adrenosterone(4-androstene-3,11,17-trione), dehydroepiandrosterone(3beta-hydroxy-5-androsten-17-one), dehydroepiandrosterone sulfate(3-beta-sulfooxy-5-androsten-17-one), testosterone(17beta-hydroxy-4-androsten-3-one), epitestosterone(17-alpha-hydroxy-4-androsten-3-one), 5-alpha-dihydrotesterone(17-beta-hydroxy-5alpha-androstan-3-one), 5-beta-dihydrotestosterone(17-beta-hydroxy-5beta-androstan-3-one), 11-beta-hydroxytesosterone(11-beta,17beta-dihydroxy-4-androsten-3-one), 11-ketotesosterone(17-beta-hydroxy-4-androsten-3,17-dione), estrogen (including: estrone(3-hydroxy-1,3,5(10)-estratrien-17-one), estradiol(1,3,5(10)-estratriene-3,17beta-diol), and estriol(1,3,5(10)-estratriene-3,16alpha,17beta-triol)), corticosterone(11-beta,21-dihydroxy-4-pregnene-3,20-dione), deoxycorticosterone(21-hydroxy-4-pregnene-3,20-dione), cortisol(11-beta,17,21-trihydroxy-4-pregnene-3,20-dione), 11-deoxycortisol(17,21-dihydroxy-4-pregnene-3,20-dione), cortisone(17,21-dihydroxy-4-pregnene-3,11,20-trione), 18-hydroxycorticosterone(11-beta,18,21-trihydroxy-4-pregnene-3,20-dione),1-alpha-hydroxycorticosterone(1-alpha,11-beta,21-trihydroxy-4-pregnene-3,20-dione), and aldosterone(18,11-hemi acetal of 11beta,21-dihydroxy-3,20-dioxo-4-pregnen-18-al).

As used herein, a “Specific Binding Peptide” includes an“anti-angiogenic peptide” (SEQ. ID. 146) and an “integrin bindingpeptide” (SEQ. ID. 147). A “Specific Binding Peptide” includes integrinbinding peptide RGD4C=CDCRGDFC (SEQ. ID. 147), integrin binding peptideRGD10 (SEQ. ID. 148), c(RGDyK) (SEQ. ID. 149), integrin binding peptidec(RGDfK) (SEQ. ID. 150), integrin binding peptide [c(RGDyK)]2 (SEQ. ID.151), integrin binding peptide CAGKNFFWKTFTSC (SEQ. ID. 152),cilengitide (cyclic RGD pentapeptide) (SEQ. ID. 153), ATN-161 (peptideantagonist of integrin alpha5beta1) (SEQ. ID. 154), ATN-454(Ac-PHSCN-NH₂) (peptide antagonist of integrin alpha5beta1) (SEQ. ID.155), tumstatin T7 peptide TMPFLFCNVNDVCNFASRNDYSYWL (SEQ. ID. 156),tumstatin sequence 1 YSNS (SEQ. ID. 157), tumstatin sequence 2 YSNSG(SEQ. ID. 158), endostatin motif FLSSRLQDLYSIVRRADRAA (SEQ. ID. 159),endostatin motif IVRRADRAAVP (SEQ. ID. 160), laminin peptide A13(RQVFQVAYIIIKA) (SEQ. ID. 161), laminin peptide C16 (KAFDITYVRLKF) (SEQ.ID. 162), laminin peptide C16S (DFKLFAVTIKYR) (SEQ. ID. 163), and VEGFR1peptide (CPQPRPLC) (SEQ. ID. 164).

As used herein, a traditional linker includes linkers that can be formedfrom those reagents disclosed in Tables IA-ID, IIA-IID, IIIA-IIIC,IVA-IVC, VA-VB, and VIA-VID.

As used herein, a “FSB linker” includes those linkers selected from thegroup consisting of 4-fluorosulfonyl benzoyl, 3-fluorosulfonyl benzoyland 2-fluorosulfonyl benzoyl as depicted in FIG. 15.

As used herein, a “Mall” linker includes a malonic linker and amaleimide linker covalently attached to an Illudin, Syn-Illudin, orAcylfulvene.

As used herein, a “protease” includes those enzymes disclosed in TableIX.

As used herein, a “cytokine” includes chemokines, interferons,interleukins, lymphokines, tumor necrosis factor, neutrophil activatingprotein-2, and monocyte chemotactic protein-1 and those compoundsdisclosed in Table XIV.

Despite recent advances in therapy, many patients with cancer invariablyrelapse and require additional treatments. Most of these patient'scancers become refractory to standard chemotherapy and/or radiationtreatment regimens. The prognosis for these patients is poor and longterm survival rates for metastatic solid tumor cancers remain very low.Thus, there is a need for the development of novel agents and treatmentregimens that specifically target these recurring tumor cells and alsoproduce less systemic toxicity. Target therapies, such as monoclonalantibodies, now provide a promising alternative to the conventionalcytotoxic chemotherapy approach.

Monoclonal antibody based therapy has recently achieved considerablesuccess in oncology and there are currently nine monoclonal antibodies(without a medicant attached) approved by the FDA as cancertherapeutics. As an example, HERCEPTIN® and RITUXAN® (both produced byGenentech, South San Francisco, Calif.), are used to successfully treatbreast cancer and non-Hodgkin's lymphoma, respectively. HERCEPTIN® is arecombinant DNA-derived humanized monoclonal antibody selectivelybinding to the extracellular domain of the Human Epidermal growth factorReceptor 2 (HER2) proto-oncogene whereas RITUXAN® is a geneticallyengineered chimeric murine/human monoclonal antibody directed againstthe CD20 antigen overexpressed on the surface of normal and malignant Blymphocytes.

Recent clinical evidence indicates that while the monoclonal antibodybased therapies are effective at inducing remission, they do not alwaysproduce a complete cure, and relapses eventually occur in most patients.There is now a tremendous interest in the use of antibody medicantconjugates as a class of therapeutics that utilize theantigen-selectivity of monoclonal antibodies to deliver potent cytotoxicmedicants to specific tumor cells. Antibody medicant conjugates areproduced by attaching a cytotoxic agent to an antibody that bindsspecifically to a tumor-associated antigen.

In theory, antibody medicant conjugates can confer an increasedtherapeutic index to highly potent medicants by improving therapeuticefficacy and reducing systemic toxicity (by minimizing damage to normaltissues), although this goal has been elusive in achieving. The basisfor the efficacy of antibody medicant conjugates is that they targettumor cells that preferentially express an antigen that is recognized bythe associated antibody. In contrast, non-tumor cells either fail toexpress this antigen, or express the antigen at a very low level. Intheory, only the tumor cells expressing the associated antibody arerecognized and destroyed by the AMC, and other cells are left untouchedand undamaged.

While different medicant classes have been tried for delivery viaantibodies, only a few have proved efficacious for use as antibodymedicant conjugates. The two main medicant classes used to date toproduce antibody medicant conjugates are the auristatins(MMAE/N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine orMMAF/N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine) andthe maytansines (DM1 or DM4). Currently only two antibody medicantconjugates are approved by the U.S.F.D.A. and marketed; brentuximabvedotin (auristatin based) and ado-trastuzumab emtansine (maytansinebased).

Illudins (see FIG. 20A, where R=CH₃OH or OH), Syn-illudins (see FIG.20B, where X or Y=C, N, S, O and Z═O or NH or NOH), and Acylfulvenes(see FIG. 20C and FIG. 20D, where X=C, N, S, O and n>1) have severalunique properties over agents traditional used to make antibody drugconjugates (ADCs). Firstly, these are the only agents known to functionby inhibition of the DNA transcription-coupled repair pathway (see FIG.5). No other toxin, drug or medicant inhibits this pathway. The resultis that Illudins, Syn illudins, and Acylfulvenes are true cytotoxicagents whereas other agents traditionally used to produce ADCs(pyrrolobenzodiazepines, maytansines, fumagillols, dolstatins,auristatins, enadiynes, halichondrins, and tubulysins) are onlycytostatic. In the NCI-DTP 60 cell line panel these other agents werecapable of inhibiting tumor cell growth (IC₅₀ value), had some abilityto block tumor cell growth (TGI value) but none were capable of actuallycausing tumor cell death or cytotoxicity (Table XIII). The illudinderivatives, however, are capable of killing tumor cells at nanomolarconcentrations (Table XIII). This means that while ADCs developed usingother toxins can stall tumor cell growth, they cannot actually kill thetumor cell. Once the effect of the drug has worn off the tumor cellswill again grow and kill the patient. In contrast, the Illudins,Syn-illudins, and Acylfulvenes actually kill the tumor cell with aslittle as a 2 hour exposure (see FIG. 4). Secondly, whereas tumor cellswill undergo apoptosis or cell death with hours once the DNAtranscription-coupled repair pathway is blocked, normal diploidnon-tumor cells can survive for hours. This translates into a widetherapeutic window for ADCs developed with Illudins, Syn-illudins, andAcylfulvenes. The two ADC agents currently FDA approved foradministration deliver a dose of the associated toxin that is 300%higher than a lethal dose which is why these agents have severe systemictoxicity. In contrast, the comparable ADC developed with Illudins,Syn-illudins, or Acylfulvenes will deliver a dose of the associatedtoxin that is 40% of a known non-toxic dose (estimated at 28% of a toxicdose and only 12% of a lethal dose). Thus, ADCS developed with Illudins,Syn-illudins, and Acylfulvenes will have minimal systemic toxicity ascompared to current agents. Thirdly, these agents are stable down to apH of 2.0. An ADC is engulfed by a tumor cell, transported to theendosomes (pH<6.0) and then into the lysozomes (pH<4). Many agents usedfor ADCs will degrade in these low pH environment, whereas Illudins,Syn-illudins, and Acylfulvenes are stable. 4) Cancer cells can becomeresistant to various toxins and drugs through the development of what istermed multi-drug resistance. This process is known to occur throughseveral different mechanisms. Whereas other toxins and drugs aresubstrates for the most common MDR mechanisms (MDR1/gp170 andMRP/gp180), and cancer cells can become resistant to these agents, theIlludins, Syn-illudins, and Acylfulvenes remain active against all MDRphenotypes regardless of the mechanism (see FIG. 7 and Table XIV).Hence, if tumor cells have already developed multi-drug resistance priorto ADC with a conventional toxin, or during the administration of acourse of the ADC, the ADC will have no efficacy. In contrast, ADCsdeveloped with Illudins, Syn-illudins, or Acylfulvenes will continue tokill cancer cells.

The present invention is based on the unexpected discovery thatacylfulvenes, can be conjugated directly to a linker, via a variety ofpeptide or non-peptide bonds, and are active as medicant delivery agentsin vitro and in vivo. Similar to other medicant classes used to produceantibody medicant conjugates, the acylfulvenes can be conjugated to alinker that allows subsequent coupling to a monoclonal antibody.Surprisingly, unlike previous medicant classes such as the auristatins(MMAE, MMAF, dolstatin-10), the maytansines (DM1 or DM4), theirinotecans and their metabolites (SN38), the calicheamicins (17-DMAG),the pyrrolobenzodiazepines (SJG-136), the duocarmycins (CC-1065), manyof the acylfulvene compounds do not require a linker and can be directlyattached to a monoclonal antibody or fragment thereof by a variety ofsimple chemical reactions. In this sense, the lack of requirement for alinker or a spacer, the acylfulvene compounds are unique. They willdirectly form covalent bonds with reactive groups on an AM such as amonoclonal antibody. In addition, because of their very small size andextreme cytotoxicity the acylfulvenes can be coupled directly to verysmall molecular weight entities (or affinity moieties) that allow tumorspecific cytotoxicity without the concomitant requirement of use of amonoclonal antibody. Examples include the ability to linkilludins/acylfulvenes directly to steroids which allow themedicant-affinity complex to kill cells overexpressing a specificsteroid receptor (such as estrogen- or progesterone-positive breastcancer cells) or even to be chemically coupled to various lipids. Thesmall size and extreme cytotoxicity acylfulvenes allows direct couplingto peptides which can preferentially bind to tumor cells (integrinbinding peptides) or display anti-angiogenic properties to hinder tumorinvasion. The illudins/acylfulvenes can also be coupled to specificpeptides which actually renders the medicant-affinity complex non-toxicuntil the peptide is cleaved by a protease secreted by tumor cells. Anexample includes PSA (prostate specific antigen) secreted by prostateadenocarcinoma cells. Again, unlike previous medicant classes such asthe auristatins (MMAE, MMAF, dolstatin-10), the maytansines (DM1 orDM4), the irinotecans and their metabolites (SN38), the calicheamicins(17-DMAG), the pyrrolobenzodiazepines (SJG-136), the duocarmycins(CC-1065), the acylfulvene compounds do not require a linker and can bedirectly attached to a steroid or a peptide that will subsequentlyfunction as an AM and direct the associated complex to specific tumorcells. An acylfulvene is attached to either a Specific Binding Peptideor a peptide which if cleaved by a specific protease (see Table IX) suchas PSA generates an entity which is cytotoxic (see Table VIII).

Trastuzumab emtansine (Genentech for Breast cancer) uses maytanasinederive DM-1, a stable non-cleavable linker Brentuximab vedotin (SeattleGenetics/Takeda for Hodgkin's Lymphoma) uses auristatin MMAE toanti-CD30, an enzyme sensitive cleavable linker.

The malonic linker, maleimide linker and SMCC [succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate] linker can form activeintermediates that react with sulfhydryl groups on an antibody. SMCC hasbeen used to bind maytansine derivative DM1 to the monoclonal antibodyHerceptin. The AMC was internalized where the Herceptin was degraded byproteases and DM1 was released into the cytosol. Further, Sulfo-SMCC[sulfosuccinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene] formsan active intermediate that reacts with sulfhydryl groups on anantibody. The resulting Sulfo-SMCC AMC is more water soluble than theSMCC AMC.

Compounds and Conjugates. The present invention is drawn to a series ofcompounds and conjugates containing a Medicant moiety (M) linked via itsC terminus to a LU (LU). The LU can operate to provide a suitablerelease of M.

In one group of embodiments, the invention provides Medicant Linkercompounds having Formula I: LU-M (I) or a pharmaceutically acceptablesalt or solvate thereof where the medicant loading is represented by p,the average number of medicant molecules per affinity (e.g., anantibody) (e.g. of Formula II, Ha, Ha′). Medicant loading may range from1 to 20 Medicant units (M) per Affinity unit (e.g., Ab or in Ab).Compositions of Formula Ha and Formula Ha′ include mixtures ofantibodies conjugated with a range of medicants, from 1 to 20.

In some embodiments, p is from about 1 to about 8 Medicant units perAffinity unit. In some embodiments, p is 1. In some embodiments, p isfrom about 2 to about 8 Medicant units per Affinity unit. In someembodiments, p is from about 2 to about 6, 2 to about 5, or 2 to about 4Medicant units per LU. In some embodiments, p is about 2, about 4, about6 or about 8 Medicant units per Affinity unit.

The average number of Medicants units per Affinity unit in a preparationfrom a conjugation reaction may be characterized by conventional meanssuch as mass spectroscopy, ELISA assay, and HPLC. The quantitativedistribution of Affinity Medicant Linker conjugates in terms of p mayalso be determined. In some instances, separation, purification, andcharacterization of homogeneous Affinity Medicant Linker conjugates,where p is a certain value from Affinity Medicant Linker conjugates withother medicant loadings may be achieved by means such as reverse phaseHPLC or electrophoresis.

Returning to Formula Ha′, the conjugates comprise an antibody covalentlyattached to one or more Medicant units (moieties) via a LU: A, a, W andw are as described above. The antibody medicant conjugate includepharmaceutically acceptable salts or solvates thereof.

The medicant loading is represented by p, the average number of Medicantunits per antibody in a molecule of Formula II. Medicant loading mayrange from 1 to 20 medicants (M) per Ab or mAb. Compositions of the AMCof Formula Ha′ include mixtures of antibodies conjugated with a range ofmedicants, from 1 to 20. In some embodiments, p is from about 1 to about8 Medicant units per antibody. In some embodiments, p is 1. In someembodiments, p is from about 2 to about 8 Medicant units per antibody.In some embodiments, p is from about 2 to about 6, 2 to about 5, or 2 toabout 4 Medicant units per antibody. In some embodiments, p is about 2,about 4, about 6 or about 8 Medicant units per antibody.

The average number of medicants per antibody in preparations of AMCsfrom conjugation reactions may be characterized by conventional meanssuch as UV/visible spectroscopy, mass spectrometry, ELISA assay, andHPLC. The quantitative distribution of AMCs in terms of p may also bedetermined. In some instances, separation, purification, andcharacterization of homogeneous AMCs where p is a certain value from AMCwith other medicant loadings may be achieved by means such as reversephase HPLC or electrophoresis.

For some antibody medicant conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, an antibody may have only one or several cysteine thiolgroups, or may have only one or several sufficiently reactive thiolgroups through which a LU may be attached. In some embodiments, thecysteine thiol is a thiol group of a cysteine residue that forms aninterchain disulfide bond. In some embodiments, the cysteine thiol is athiol group of a cysteine residue that does not form an interchaindisulfide bond.

Typically, less than the theoretical maximums of medicant moieties areconjugated to an antibody during a conjugation reaction. An antibody maycontain, for example, many lysine residues that do not react with theMedicant Linker compound intermediate or LU reagent. Only the mostreactive lysine groups may react with an amine-reactive LU reagent.Generally, antibodies do not contain many, if any, free and reactivecysteine thiol groups which may be linked to a Medicant moiety via a LU.Most cysteine thiol residues in the antibodies exist as disulfidebridges and must be reduced with a reducing agent such as dithiothreitol(DTT). The antibody may be subjected to denaturing conditions to revealreactive nucleophilic groups such as lysine or cysteine. The loading(medicant/antibody ratio) of an AMC may be controlled in severaldifferent manners, including: (i) limiting the molar excess of MedicantLinker compound intermediate or LU reagent relative to antibody, (ii)limiting the conjugation reaction time or temperature, and (iii) partialor limiting reductive conditions for cysteine thiol modification.

Where more than one nucleophilic group reacts with a Medicant Linkercompound intermediate, or LU reagent followed by Medicant moietyreagent, then the resulting product is a mixture of Affinity MedicantLinker Conjugates (e.g., AMCs) with a distribution of one or moreMedicant moieties per Affinity unit (e.g., an antibody). The averagenumber of medicants per Affinity unit (e.g., antibody) may be calculatedfrom the mixture by, for example, dual enzyme linked immune serum assay(ELISA) antibody assay, specific for antibody and specific for themedicant. Individual Affinity Medicant Linker Conjugate molecules may beidentified in the mixture by mass spectroscopy, and separated by highperformance liquid chromatography (HPLC), e.g., hydrophobic interactionchromatography. Thus, a homogeneous conjugate with a single loadingvalue may be isolated from the conjugation mixture by electrophoresis orchromatography.

A “Linker Unit” (LU) is a bifunctional compound which can be used tolink a Medicant unit and/or an Affinity unit to form an AffinityMedicant Linker conjugate. Such conjugates are useful, for example, inthe formation of immuno conjugates directed against tumor associatedantigens. Such conjugates allow the selective delivery of cytotoxicdrugs to tumor cells. A LU includes a traditional linker, a4-fluorosulfonyl benzoyl (4-FSB) linker, a 3-fluorosulfonyl benzoyl(3-FSB) linker a 2-fluorosulfonyl benzoyl (2-FSB) linker, a maleimide(Mall) linker, an azlactone linker and a bridging amino acid.

A traditional linker is a linker as defined in Table I through Table VI,where the reagent column identifies various traditional linkers. AStretcher Unit includes two or more Linker Units.

A bridging amino acid means —NH—C(R′)H—CO— or —N(R″)—C(R′)H—CO—including glycine, L-alanine, L-serine, L-threonine, L-cysteine,L-valine, L-leucine, L-isoleucine, L-methionine, L-proline,L-phenylalanine, L-tyrosine, L-tryptophan, L-aspartic acid, L-glutamicacid, L-asparagine, L-glutamine, L-histidine, L-lysine, L-arginine,L-homocysteine, L-selenocysteine, L-pyrrolysine, L-carnitine,L-hypusine, 2-aminoisobutyric acid, dehydroalanine, L-gamma-aminobutyricacid, L-ornithine, L-citrulline, L-α-Amino-n-butyric acid, L-Norvaline,L-Norleucine, L-Pipecolic acid, L-Alloisoleucine, L-α,β-diaminopropionicacid, L-α,γ-diaminobutyric acid, L-Allothreonine, L-α-Amino-n-heptanoicacid, L-Homoserine, β-Amino-n-butyric acid, β-Aminoisobutyric acid,γ-Aminobutyric acid, L-isovaline, L-Sarcosine, N-ethyl glycine, N-propylglycine, N-isopropyl glycine, L-N-methyl alanine, L-N-ethyl alanine,N-methyl β-alanine, N-ethyl β-alanine, Isoserine,L-α-hydroxy-γ-aminobutyric acid, L-diaminopimelic acid, cystathione,L-aminoisobutyric acid, dehydroalanine, delta-aminolevulinic acid,4-aminobenzoic acid, L-Hydroxyproline, Formylmethioinine, L-lanthionine,djenkolic acid, L-Pyroglutamic acid, Hypusine, L-carboxyglutamic acid,penicillamine, L-thialysine, quisqualic acid, L-canavine,L-azetidine-2-carboxylic acid, D-alanine, D-serine, D-threonine,D-cysteine, D-valine, D-leucine, D-isoleucine, D-methionine, D-proline,D-phenylalanine, D-tyrosine, D-tryptophan, D-aspartic acid, D-glutamicacid, D-asparagine, D-glutamine, D-histidine, D-lysine, D-arginine,D-homocysteine, D-selenocysteine, D-pyrrolysine, D-carnitine,D-hypusine, D-gamma-aminobutyric acid, D-ornithine, D-citrulline,D-α-Amino-n-butyric acid, D-Norvaline, D-Norleucine, D-Pipecolic acid,D-Alloisoleucine, D-α,β-diaminopropionic acid, D-α,γ-diaminobutyricacid, D-Allothreonine, D-α-Amino-n-heptanoic acid, D-Homoserine,D-isovaline, D-Sarcosine, D-N-methyl alanine, D-N-ethyl alanine,D-α-hydroxy-γ-aminobutyric acid, D-diaminopimelic acid,D-aminoisobutyric acid, D-Hydroxyproline, D-lanthionine, D-Pyroglutamicacid, D-carboxyglutamic acid, D-thialysine, quisqualic acid, D-canavine,D-azetidine-2-carboxylic acid. A ‘modified bridging amino acid’ means abridging amino acid with R′ including a hydroxyl group that has beenesterified, a bridging amino acid with R′ including a sulphur atom wherethe sulphur atom has been reacted with an alkyl or other organic groupand/or a bridging amino acid with R′ including a primary amino groupthat has been converted into a secondary or tertiary amino group.

In one embodiment, the LU of the Medicant Linker compound and AffinityMedicant Linker conjugate has the formula: —W_(w)-A_(a) wherein -A- is aStretcher Unit; a is 1 or 2; each —W— is independently an Amino Acidunit; w is independently an integer ranging from 1 to 20. In theAffinity Medicant Linker conjugate, the LU serves to attach the Medicantmoiety and the AM.

The Affinity Moiety (AM) includes within its scope an Affinity Unit (AU)that specifically binds or reactively associates or complexes with areceptor, antigen or other receptive moiety associated with a giventarget-cell population. An AU is a molecule that binds to, complexeswith, or reacts with a receptor, antigen or other receptive moiety of acell population sought to be therapeutically or otherwise biologicallymodified. In one aspect, the AM acts to deliver the Medicant unit to theparticular target cell population with which the AM interacts. Such AM'sinclude, but are not limited to, proteins, polypeptides and peptides andinclude, antibodies, binding proteins, smaller molecular weightproteins, polypeptides, peptides, lectins, glycoproteins, non-peptides,vitamins, nutrient-transport molecules (such as, but not limited to,transferrin), or any other cell binding molecule or substance.

In an embodiment of the invention, an AM can form a bond to a StretcherUnit. In an alternative embodiment of the invention, an AM can form abond to the Stretcher Unit of the LU via a heteroatom of the AM.Heteroatoms that may be present on an AM include sulfur (in oneembodiment, from a sulfhydryl group of an AM), oxygen (in oneembodiment, from a carbonyl, carboxyl or hydroxyl group of an AM) andnitrogen (in one embodiment, from a primary or secondary amino group ofan AM). These hetero atoms can be present on the AM in the AM's naturalstate, for example a naturally-occurring antibody, or can be introducedinto the AM via chemical modification.

In one embodiment, an AM unit has a sulfhydryl group and the AM bonds tothe LU via the sulfhydryl group's sulfur atom. In another embodiment,the AM has lysine residues that can react with activated esters (suchesters include, but are not limited to, N-hydroxysuccinimide,pentafluorophenyl, and p-nitrophenyl esters) of the Stretcher Unit ofthe AM and thus form an amide bond consisting of the primary nitrogenatom of the AM and the carboxyl group of the AM. In yet another aspect,the AM has one or more lysine residues that can be chemically modifiedto introduce one or more sulfhydryl groups. The AM bonds to the LU viathe sulfhydryl group's sulfur atom. The reagents that can be used tomodify lysines include, but are not limited to, N-succinimidylS-acetylthioacetate (SATA) and 2-Iminothiolane hydrochloride (Traut'sReagent).

In another embodiment, the AM can have one or more carbohydrate groupsthat can be chemically modified to have one or more sulthydryl groups.The AM bonds to the LU (or a Stretcher Unit) via the sulfhydryl group'ssulfur atom. In yet another embodiment, the AM can have one or morecarbohydrate groups that can be oxidized to provide an aldehyde (—CHO)group. The corresponding aldehyde can form a bond with a reactive siteon a Stretcher Unit. Reactive sites on a Stretcher Unit that can reactwith a carbonyl group on an AM include, but are not limited to,hydrazine and hydroxylamine.

Useful non-immunoreactive protein, polypeptide, or peptide affinitymoieties include, but are not limited to, transferrin, epidermal growthfactors (“EGF”), bombesin, gastrin, gastrin-releasing peptide,platelet-derived growth factor, IL-2, IL-6, transforming growth factors(“TOP”), such as TGF-.alpha. and TGF-.beta., vaccinia growth factor(“VGF”), insulin and insulin-like growth factors I and II, somatostatin,lectins and apoprotein from low density lipoprotein.

Useful polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of immunized animals. Useful monoclonalantibodies are homogeneous populations of antibodies to a particularantigenic determinant (e.g., a cancer cell antigen, a viral antigen, amicrobial antigen, a protein, a peptide, a carbohydrate, a chemical,nucleic acid, or fragments thereof). A monoclonal antibody (mAb) to anantigen-of-interest can be prepared by using any technique known in theart which provides for the production of antibody molecules bycontinuous cell lines in culture.

Useful monoclonal antibodies include, but are not limited to, humanmonoclonal antibodies, humanized monoclonal antibodies, antibodyfragments, or chimeric monoclonal antibodies. Human monoclonalantibodies may be made by any of numerous techniques known in the art.

The antibody can also be a bispecific antibody. Methods for makingbispecific antibodies are known in the art and are discussed infra.

The antibody can be a functionally active fragment, derivative or analogof an antibody that immunospecifically binds to target cells (e.g.,cancer cell antigens, viral antigens, or microbial antigens) or otherantibodies that bind to tumor cells or matrix. In this regard,“functionally active” means that the fragment, derivative or analog isable to elicit anti-anti-idiotype antibodies that recognize the sameantigen that the antibody from which the fragment, derivative or analogis derived recognized. Specifically, in an exemplary embodiment theantigenicity of the idiotype of the immunoglobulin molecule can beenhanced by deletion of framework and CDR sequences that are C-terminalto the CDR sequence that specifically recognizes the antigen. Todetermine which CDR sequences bind the antigen, synthetic peptidescontaining the CDR sequences can be used in binding assays with theantigen by any binding assay method known in the art (e.g., the BIA coreassay).

Other useful antibodies include fragments of antibodies such as, but notlimited to, F(ab′)₂ fragments, Fab fragments, Fvs, single chainantibodies, diabodies, triabodies, tetrabodies, scFv, scFv-FV, or anyother molecule with the same specificity as the antibody.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are usefulantibodies. A chimeric antibody is a molecule in which differentportions are derived from different animal species, such as for example,those having a variable region derived from a murine monoclonal andhuman immunoglobulin constant regions. Humanized antibodies are antibodymolecules from non-human species having one or more complementaritydetermining regions (CDRs) from the non-human species and a frameworkregion from a human immunoglobulin molecule. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art.

Completely human antibodies are particularly desirable and can beproduced using transgenic mice that are incapable of expressingendogenous immunoglobulin heavy and light chains genes, but which canexpress human heavy and light chain genes. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. Other human antibodies can be obtainedcommercially from, for example, Abgenix, Inc. (now Amgen, Freemont,Calif.) and Medarex (Princeton, N.J.).

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. Human antibodies can also be producedusing various techniques known in the art, including phage displaylibraries.

In other embodiments, the antibody is a fusion protein of an antibody,or a functionally active fragment thereof, for example in which theantibody is fused via a covalent bond (e.g., a peptide bond), at eitherthe N-terminus or the C-terminus to an amino acid sequence of anotherprotein (or portion thereof, preferably at least 10, 20 or 50 amino acidportion of the protein) that is not from an antibody. Preferably, theantibody or fragment thereof is covalently linked to the other proteinat the N-terminus of the constant domain.

Antibodies include analogs and derivatives that are either modified,i.e., by the covalent attachment of any type of molecule as long as suchcovalent attachment permits the antibody to retain its antigen bindingimmunospecificity. For example, but not by way of limitation,derivatives and analogs of the antibodies include those that have beenfurther modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular antibody unit orother protein, etc. Any of numerous chemical modifications can becarried out by known techniques including, but not limited to, specificchemical cleavage, acetylation, formylation, metabolic synthesis in thepresence of tunicamycin, etc. Additionally, the analog or derivative cancontain one or more unnatural amino acids.

Antibodies can have modifications (e.g., substitutions, deletions oradditions) in amino acid residues that interact with Fc receptors. Inparticular, antibodies can have modifications in amino acid residuesidentified as involved in the interaction between the anti-Fc domain andthe FcRn receptor.

Antibodies immunospecific for a cancer cell antigen can be obtainedcommercially or produced by any method known to one of skill in the artsuch as, e.g., chemical synthesis or recombinant expression techniques.The nucleotide sequence encoding antibodies immunospecific for a cancercell antigen can be obtained, e.g., from the GenBank database or adatabase like it, literature publications, or by routine cloning andsequencing.

In a specific embodiment, known antibodies for the treatment of cancercan be used. Antibodies immunospecific for a cancer cell antigen can beobtained commercially or produced by any method known to one of skill inthe art such as, e.g., recombinant expression techniques. The nucleotidesequence encoding antibodies immunospecific for a cancer cell antigencan be obtained, e.g., from the GenBank database or a database like it,the literature publications, or by routine cloning and sequencing.Examples of antibodies available for the treatment of cancer include,but are not limited to, RITUXAN® (rituximab; Genentech) which is achimeric anti-CD20 monoclonal antibody for the treatment of patientswith non-Hodgkin's lymphoma; OVAREX which is a murine antibody for thetreatment of ovarian cancer; PANOREX (Glaxo Wellcome, N.C.) which is amurine IgG_(2a) antibody for the treatment of colorectal cancer;Cetuximab ERBITUX (Imclone Systems Inc., NY) which is an anti-EGFR IgGchimeric antibody for the treatment of epidermal growth factor positivecancers, such as head and neck cancer; Vitaxin (MedImmune, Inc., MD)which is a humanized antibody for the treatment of sarcoma; CAMPATH I/H(Leukosite, MA) which is a humanized IgG₁ antibody for the treatment ofchronic lymphocytic leukemia (CLL); SMART MI95 (Protein Design Labs,Inc., CA) and SGN-33 (Seattle Genetics, Inc., WA) which is a humanizedanti-CD33 IgG antibody for the treatment of acute myeloid leukemia(AML); LYMPHOCIDE (Immunomedics, Inc., NJ) which is a humanizedanti-CD22 IgG antibody for the treatment of non-Hodgkin's lymphoma;SMART ID10 (Protein Design Labs, Inc., CA) which is a humanizedanti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma;ONCOLYM (Techniclone, Inc., CA) which is a radiolabeled murineanti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma;ALLOMUNE (BioTransplant, CA) which is a humanized anti-CD2 mAb for thetreatment of Hodgkin's Disease or non-Hodgkin's lymphoma; AVASTIN(Genentech, Inc., CA) which is an anti-VEGF humanized antibody for thetreatment of lung and colorectal cancers; Epratuzamab (Immunomedics,Inc., NJ and Amgen, Calif.) which is an anti-CD22 antibody for thetreatment of non-Hodgkin's lymphoma; and CEACIDE (Immunomedics, N.J.)which is a humanized anti-CEA antibody for the treatment of colorectalcancer.

Other antibodies useful in the treatment of cancer include, but are notlimited to, antibodies against the following antigens (where exemplarycancers that can be treated with the antibody are in parentheses): Alk(adrenocarcinomas) (SEQ. ID. 103), CA125 (ovarian) (SEQ. ID. 104),CA15-3 (carcinomas) (SEQ. ID. 105), CA19-9 (carcinomas), L6 (carcinomas)(SEQ. ID. 107), Lewis Y (carcinomas) (SEQ. ID. 108), Lewis X(carcinomas) (SEQ. ID. 109), alpha fetoprotein (carcinomas) (SEQ. ID.110), CA 242 (colorectal), placental alkaline phosphatase (carcinomas)(SEQ. ID. 112), prostate specific antigen (prostate) (SEQ. ID. 113),prostate specific membrane antigen (prostate) (SEQ. ID. 114), prostaticacid phosphatase (prostate) (SEQ. ID. 115), epidermal growth factor(carcinomas), MAGE-1 (carcinomas) (SEQ. ID. 117), MAGE-2 (carcinomas)(SEQ. ID. 118), MAGE-3 (carcinomas) (SEQ. ID. 119), MAGE-4 (carcinomas)(SEQ. ID. 120), anti-transferrin receptor (carcinomas) (SEQ. ID. 121),p97 (melanoma) (SEQ. ID. 122), MUC1 (breast cancer) (SEQ. ID. 123), CEA(colorectal) (SEQ. ID. 124), gp100 (melanoma) (SEQ. ID. 125), MART-1(melanoma) (SEQ. ID. 126), IL-2 receptor (T-cell leukemia andlymphomas), CD2 (buccal mucosa) (SEQ. ID. 128), CD20 (non-Hodgkin'slymphoma) (SEQ. ID. 129), CD52 (leukemia) (SEQ. ID. 130), CD33(leukemia), CD22 (lymphoma), beta human chorionic gonadotropin(carcinoma) (SEQ. ID. 133), CD38 (multiple myeloma) (SEQ. ID. 134), CD40(lymphoma) (SEQ. ID. 135), CD80 (colorectal), CD86 (colorectal), mucin(carcinomas), P21 (carcinomas), MPG (melanoma) (SEQ. ID. 140), Neuoncogene product (carcinomas) and STEAP-1 (prostate).

Compositions and Methods of Administration. In other embodiments,described is a pharmaceutical composition including an effective amountof an Affinity Medicant Linker conjugate and/or a Medicant Linkercompound and a pharmaceutically acceptable carrier or vehicle. Thecompositions are suitable for veterinary or human administration.

The present pharmaceutical compositions can be in any form that allowsfor the composition to be administered to a patient. For example, thecomposition can be in the form of a solid or liquid. Typical routes ofadministration include, without limitation, parenteral, ocular andintra-tumor. Parenteral administration includes subcutaneous injections,intravenous, intramuscular or intrasternal injection or infusiontechniques. In one aspect, the compositions are administeredparenterally. In a specific embodiment, the compositions areadministered intravenously.

Pharmaceutical compositions can be formulated so as to allow an AffinityMedicant Linker conjugate and/or a Medicant Linker compound to bebioavailable upon administration of the composition to a patient.Compositions can take the form of one or more dosage units, where forexample, a tablet can be a single dosage unit, and a container of anAffinity Medicant Linker conjugate and/or a Medicant Linker compound inliquid form can hold a plurality of dosage units.

Materials used in preparing the pharmaceutical compositions can benon-toxic in the amounts used. It will be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of animal (e.g.,human), the particular form of the Affinity Medicant Linker conjugateand/or a Medicant Linker compound, the manner of administration, and thecomposition employed.

The pharmaceutically acceptable carrier or vehicle can be solid orparticulate, so that the compositions are, for example, in tablet orpowder form. The carrier(s) can be liquid. In addition, the carrier(s)can be particulate.

The composition can be in the form of a liquid, e.g., a solution,emulsion or suspension. In a composition for administration byinjection, one or more of a surfactant, preservative, wetting agent,dispersing agent, suspending agent, buffer, stabilizer and isotonicagent can also be included.

The liquid compositions, whether they are solutions, suspensions orother like form, can also include one or more of the following: sterilediluents such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils such as synthetic mono or digylcerides which can serve as thesolvent or suspending medium, polyethylene glycols, glycerin,cyclodextrin, propylene glycol or other solvents; antibacterial agentssuch as benzyl alcohol or methyl paraben; antioxidants such as ascorbicacid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates,phosphates or amino acids and agents for the adjustment of tonicity suchas sodium chloride or dextrose. A parenteral composition can be enclosedin ampoule, a disposable syringe or a multiple-dose vial made of glass,plastic or other material. Physiological saline is an exemplaryadjuvant. An injectable composition is preferably sterile.

The amount of the Affinity Medicant Linker conjugate and/or a MedicantLinker compound that is effective in the treatment of a particulardisorder or condition will depend on the nature of the disorder orcondition, and can be determined by standard clinical techniques. Inaddition, in vitro or in vivo assays can optionally be employed to helpidentify optimal dosage ranges. The precise dose to be employed in thecompositions will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.

The compositions comprise an effective amount of an Affinity MedicantLinker conjugate and/or a Medicant Linker compound such that a suitabledosage will be obtained. Typically, this amount is at least about 0.01%of an Affinity Medicant Linker conjugate and/or a Medicant Linkercompound by weight of the composition. In an exemplary embodiment,pharmaceutical compositions are prepared so that a parenteral dosageunit contains from about 0.01% to about 2% by weight of the AffinityMedicant Linker conjugate and/or a Medicant Linker compound.

For intravenous administration, the composition can comprise from about0.01 to about 100 mg of an Affinity Medicant Linker conjugate and/or aMedicant Linker compound per kg of the patient's body weight. In oneaspect, the composition can include from about 1 to about 100 mg of anAffinity Medicant Linker conjugate and/or a Medicant Linker compound perkg of the patient's body weight. In another aspect, the amountadministered will be in the range from about 0.1 to about 25 mg/kg ofbody weight of the Affinity Medicant Linker conjugate and/or a MedicantLinker compound.

Prior art ADC's such as Kadcyla or Adcetris deliver a dose of theassociated toxin (auristatins MMAE or emtansine DM-1) that is three ormore times the lethal dose (for that toxin) which results in severesystemic (or non-target) toxicity. In contrast, Illudin and AcylfulveneADC's (such as analog 189, analog 190), analog 217, analog 218, analog219, analog 222, or analog 316 deliver less than one third (i.e., <⅓) ofa lethal dose, minimizing the risk and severity of systemic toxicity.Illudins and Acylfulvenes are true cytotoxic agents whereas other toxicagents used in prior art ADC's (e.g., pyrrolobenzodiazepines,maytansines, fumagillols, dolstatins, auristatins, enadiynes,halichondrins, and tubulysins) are only cytostatic. See Table XIII (theNCI-DTP 60 cell line table). Hence, other payloads, such as those usedin Herceptin, Adcetris or Rituxin only stall tumor cell growth and donot actually kill the tumor cells. Other payloads (e.g.,pyrrolobenzodiazepines, maytansines, fumagillols, dolstatins,auristatins, enadiynes, halichondrins, and tubulysins) are not activeagainst multidrug phenotypes, notably the MDR1/GP170 and MRP/GP180transport mechanisms (see Table XIV). Illudins and Acylfulvenes show theexcellent effect of remaining active against all MDR phenotypes knownregardless of the mechanism of resistance (see Table XIV). Hence, iftumor cells have already developed multi-drug resistance to a prior artADC with a prior art toxin, or develop multi-drug resistance during theadministration of a course of the prior art ADC with a prior art toxin,then the ADC will have no efficacy. In contrast, ADCs developed withIlludins, Syn-illudins, or Acylfulvenes have the advantageous effectthat they will continue to kill cancer cells.

Generally, the dosage of an Affinity Medicant Linker conjugate and/or aMedicant Linker compound administered to a patient is typically about0.01 mg/kg to about 20 mg/kg of the patient's body weight. In oneaspect, the dosage administered to a patient is between about 0.01 mg/kgto about 10 mg/kg of the patient's body weight. In another aspect, thedosage administered to a patient is between about 0.1 mg/kg and about 10mg/kg of the patient's body weight. In yet another aspect, the dosageadministered to a patient is between about 0.1 mg/kg and about 5 mg/kgof the patient's body weight. In yet another aspect the dosageadministered is between about 0.1 mg/kg to about 3 mg/kg of thepatient's body weight. In yet another aspect, the dosage administered isbetween about 1 mg/kg to about 3 mg/kg of the patient's body weight.

The Affinity Medicant Linker conjugate and/or a Medicant Linker compoundcan be administered by any convenient route, for example by infusion orbolus injection. Administration can be systemic or local. Variousdelivery systems are known, e.g., encapsulation in liposomes,micro-particles, microcapsules, capsules, etc., and can be used toadminister an Affinity Medicant Linker conjugate and/or a MedicantLinker compound. In certain embodiments, more than one Affinity MedicantLinker conjugate and/or a Medicant Linker compound is administered to apatient.

In specific embodiments, it can be desirable to administer one or moreAffinity Medicant Linker conjugates and/or a Medicant Linker compoundlocally to the area in need of treatment. This can be achieved, forexample, and not by way of limitation, by local infusion during surgery;topical application, e.g., in conjunction with a wound dressing aftersurgery; by injection; by means of a catheter; or by means of animplant, the implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as sialastic membranes, or fibers.In one embodiment, administration can be by direct injection at the site(or former site) of a cancer, tumor or neoplastic or pre-neoplastictissue. In another embodiment, administration can be by direct injectionat the site (or former site) of a manifestation of an autoimmunedisease.

In yet another embodiment, the Affinity Medicant Linker conjugate and/ora Medicant Linker compound can be delivered in a controlled releasesystem, such as but not limited to, a pump or various polymericmaterials can be used. In yet another embodiment, a controlled-releasesystem can be placed in proximity of the target of the Linker Affinityconjugate and/or a Medicant Linker compound, e.g., the liver, thusrequiring only a fraction of the systemic dose.

The term “carrier” refers to a diluent, adjuvant or excipient, withwhich an Affinity Medicant Linker conjugate and/or a Medicant Linkercompound is administered. Such pharmaceutical carriers can be liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin. The carriers can be saline, and the like. Inaddition, auxiliary, stabilizing and other agents can be used. In oneembodiment, when administered to a patient, the Affinity Medicant Linkerconjugate and/or the Medicant Linker compound and pharmaceuticallyacceptable carriers are sterile. Water is an exemplary carrier when theAffinity Medicant Linker conjugate and/or a Medicant Linker compound areadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. The present compositions, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents.

The present compositions can take the form of solutions, pellets,powders, sustained-release formulations, or any other form suitable foruse.

In an embodiment, the Affinity Medicant Linker conjugates and/orMedicant Linker compounds are formulated in accordance with routineprocedures as a pharmaceutical composition adapted for intravenousadministration to animals, particularly human beings. Typically, thecarriers or vehicles for intravenous administration are sterile isotonicaqueous buffer solutions. Where necessary, the compositions can alsoinclude a solubilizing agent. Compositions for intravenousadministration can optionally comprise a local anesthetic such aslignocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where an AffinityMedicant Linker conjugate and/or Medicant Linker compound is to beadministered by infusion, it can be dispensed, for example, with aninfusion bottle containing sterile pharmaceutical grade water or saline.Where the Affinity Medicant Linker conjugate and/or Medicant Linkercompound is administered by injection, an ampoule of sterile water forinjection or saline can be provided so that the ingredients can be mixedprior to administration.

The composition can include various materials that modify the physicalform of a solid or liquid dosage unit. For example, the composition caninclude materials that form a coating shell around the activeingredients. The materials that form the coating shell are typicallyinert, and can be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients can beencased in a gelatin capsule.

Whether in solid or liquid form, the present compositions can include apharmacological agent used in the treatment of cancer, an autoimmunedisease or an infectious disease.

Treatment of Cancer. The Affinity Medicant Linker conjugates andMedicant Linker compounds are useful for inhibiting the multiplicationof a tumor cell or cancer cell, causing apoptosis in a tumor or cancercell, or for treating cancer in a patient. The Affinity Medicant Linkerconjugates and/or Medicant Linker compounds can be used accordingly in avariety of settings for the treatment of animal cancers. The AffinityMedicant Linker Conjugates can be used to deliver a Medicant or Medicantunit to a tumor cell or cancer cell. Without being bound by theory, inone embodiment, the AM of an Affinity Medicant Linker conjugate binds toor associates with a cancer-cell or a tumor-cell-associated antigen, andthe Affinity Medicant Linker conjugate can be taken up (internalized)inside a tumor cell or cancer cell through receptor-mediated endocytosisor other internalization mechanism. The antigen can be attached to atumor cell or cancer cell or can be an extracellular matrix proteinassociated with the tumor cell or cancer cell. Once inside the cell, oneor more specific peptide sequences within or at the Medicant unit'sproximal end of the LU are hydrolytically cleaved by one or more tumorcell or cancer cell-associated proteases, resulting in release of theMedicant unit. The released Medicant unit is then free to migrate withinthe cell and induce cytotoxic or cytostatic activities. The AffinityMedicant Linker conjugate also can be cleaved by an intracellularprotease to release the Medicant moiety. In an alternative embodiment,the Medicant or Medicant unit is cleaved from the Affinity MedicantLinker conjugate outside the tumor cell or cancer cell, and the Medicantor Medicant unit subsequently penetrates the cell.

The Affinity Medicant Linker conjugates provide conjugation-specifictumor or cancer medicant targeting, thus reducing general toxicity ofthe Medicant. The LUs stabilize the Affinity Medicant Conjugates inblood, yet are cleavable by tumor-specific proteases within the cell,liberating a Medicant unit.

In one embodiment, the AM binds to the tumor cell or cancer cell. Inanother embodiment, the AM binds to a tumor cell or cancer cell antigenwhich is on the surface of the tumor cell or cancer cell. In anotherembodiment, the AM binds to a tumor cell or cancer cell antigen which isan extracellular matrix protein associated with the tumor cell or cancercell.

The specificity of the AM for a particular tumor cell or cancer cell canbe important for determining those tumors or cancers that are mosteffectively treated. For example, an Affinity Medicant Linker conjugateand/or Medicant Linker compound having a BR96 AM can be useful fortreating antigen positive carcinomas including those of the lung,breast, colon, ovaries, and pancreas. Affinity Medicant Linkerconjugates having an anti-CD30 or an anti-CD70 binding affinity moietycan be useful for treating hematologic malignancies.

Other particular types of cancers that can be treated with an AffinityMedicant Linker conjugate and/or a Medicant Linker compound include, butare not limited to fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer,pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostatecancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer,throat cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicularcancer, small cell lung carcinoma, bladder carcinoma, lung cancer,epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skincancer, melanoma, neuroblastoma, retinoblastoma blood-borne cancers,including but not limited to: acute lymphoblastic leukemia “ALL”, acutelymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,acute myeloblastic leukemia “AML”, acute promyelocytic leukemia “APL”,acute monoblastic leukemia, acute erythroleukemic leukemia, acutemegakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronicmyelocytic leukemia “CML”, chronic lymphocytic leukemia “CLL”, hairycell leukemia, multiple myeloma acute and chronic leukemias:lymphoblastic, myelogenous, lymphocytic, myelocytic leukemias Lymphomas:Hodgkin's disease, non-Hodgkin's Lymphoma, Multiple myeloma,Waldenstrom's macroglobulinemia, Heavy chain disease, Polycythemia vera.

Multi-Modality Therapy for Cancer. Cancers, including, but not limitedto, a tumor, metastasis, or other disease or disorder characterized byuncontrolled cell growth, can be treated or inhibited by administrationof an Affinity Medicant Linker conjugate or Medicant Linker compound.

In other embodiments, methods for treating cancer are provided,including administering to a patient in need thereof an effective amountof an Affinity Medicant Linker conjugate and a chemotherapeutic agent.In one embodiment the chemotherapeutic agent is that with whichtreatment of the cancer has not been found to be refractory. In anotherembodiment, the chemotherapeutic agent is that with which the treatmentof cancer has been found to be refractory. The Affinity Medicant Linkerconjugates can be administered to a patient that has also undergonesurgery as treatment for the cancer.

In some embodiments, the patient also receives an additional treatment,such as radiation therapy. In a specific embodiment, the AffinityMedicant Linker conjugate is administered concurrently with thechemotherapeutic agent or with radiation therapy. In another specificembodiment, the chemotherapeutic agent or radiation therapy isadministered prior or subsequent to administration of an AffinityMedicant Linker conjugate.

A chemotherapeutic agent can be administered over a series of sessions.Any one or a combination of the chemotherapeutic agents, such a standardof care chemotherapeutic agent(s), can be administered.

Additionally, methods of treatment of cancer with an Affinity MedicantLinker conjugate and/or a Medicant Linker compound are provided as analternative to chemotherapy or radiation therapy where the chemotherapyor the radiation therapy has proven or can prove too toxic, e.g.,results in unacceptable or unbearable side effects, for the subjectbeing treated. The patient being treated can, optionally, be treatedwith another cancer treatment such as surgery, radiation therapy orchemotherapy, depending on which treatment is found to be acceptable orbearable.

The Affinity Medicant Linker (AML) conjugates and/or Medicant Linker(ML) compounds can also be used in an in vitro or ex vivo fashion, suchas for the treatment of certain cancers, including, but not limited toleukemia and lymphomas, such treatment involving autologous stem celltransplants. This can involve a multi-step process in which the animal'sautologous hematopoietic stein cells are harvested and purged of allcancer cells, the animal's remaining bone-marrow cell population is theneradicated via the administration of a high dose of an AML conjugatesand/or ML compound with or without accompanying high dose radiationtherapy, and the stem cell graft is infused back into the animal.Supportive care is then provided while bone marrow function is restoredand the patient recovers.

Treatment of Autoimmune Diseases. The Affinity Medicant Linkerconjugates and Medicant Linker compounds are useful for killing orinhibiting the replication of a cell that produces an autoimmune diseaseor for treating an autoimmune disease. The Affinity Medicant Linkerconjugates and Medicant Linker compounds can be used accordingly in avariety of settings for the treatment of an autoimmune disease in apatient. The Affinity Medicant Linker conjugates can be used to delivera Medicant unit to a target cell. Without being bound by theory, in oneembodiment, the Affinity Medicant Linker conjugate associates with anantigen on the surface of a target cell, and the Affinity MedicantLinker conjugate is then taken up inside a target-cell throughreceptor-mediated endocytosis. Once inside the cell, one or morespecific peptide sequences within and/or Medicant unit proximal to theLU are enzymatically or hydrolytically cleaved, resulting in release ofthe Medicant or Medicant unit. The released Medicant or Medicant unit isthen free to migrate in the cytosol and induce cytotoxic or cytostaticactivities. The Affinity Medicant Linker conjugate also can be cleavedby an intracellular protease to release the Medicant or Medicant moiety.In an alternative embodiment, the Medicant is cleaved from the AffinityMedicant Linker conjugate outside the target cell, and the Medicant orMedicant unit subsequently penetrates the cell.

In an embodiment of the present invention, the AM binds to an autoimmuneantigen. In one aspect, the antigen is on the surface of a cell involvedin an autoimmune condition. In another embodiment, the AM binds to anautoimmune antigen which is on the surface of a cell. In one embodiment,the AM binds to activated lymphocytes that are associated with theautoimmune disease state.

In a further embodiment, the Affinity Medicant Linker conjugate orMedicant Linker compound kills or inhibit the multiplication of cellsthat produce an autoimmune antibody associated with a particularautoimmune disease.

In various embodiments of the present invention, the AML or AMconjugates can be used to treat particular types of autoimmune diseasesincluding, but not limited to, Th2 lymphocyte related disorders (e.g.,atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergicrhinitis, Omenn's syndrome, systemic sclerosis, and graft versus hostdisease); Thi lymphocyte-related disorders (e.g., rheumatoid arthritis,multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto'sthyroiditis, Grave's disease, primary biliary cirrhosis, Wegener'sgranulomatosis, and tuberculosis); activated B lymphocyte-relateddisorders (e.g., systemic lupus erythematosus, Goodpasture's syndrome,rheumatoid arthritis, and type I diabetes); and Active ChronicHepatitis, Addison's Disease, Allergic Alveolitis, Allergic Reaction,Allergic Rhinitis, Alport's Syndrome, Anaphlaxis, AnkylosingSpondylitis, Anti-phosholipid Syndrome, Arthritis, Ascariasis,Aspergillosis, Atopic Allergy, Atropic Dermatitis, Atropic Rhinitis,Behcet's Disease, Bird-Fancier's Lung, Bronchial Asthma, Caplan'sSyndrome, Cardiomyopathy, Celiac Disease, Chagas' Disease, ChronicGlomerulonephritis, Cogan's Syndrome, Cold Agglutinin Disease,Congenital Rubella Infection, CREST Syndrome, Crohn's Disease,Cryoglobulinemia, Cushing's Syndrome, Dermatomyositis, Discoid Lupus,Dressler's Syndrome, Eaton-Lambert Syndrome, Echovirus Infection,Encephalomyelitis, Endocrine opthalmopathy, Epstein-Barr VirusInfection, Equine Heaves, Erythematosis, Evan's Syndrome, Felty'sSyndrome, Fibromyalgia, Fuch's Cyclitis, Gastric Atrophy,Gastrointestinal Allergy, Giant Cell Arteritis, Glomerulonephritis,Goodpasture's Syndrome, Graft v. Host Disease, Graves' Disease,Guillain-Barre Disease, Hashimoto's Thyroiditis, Hemolytic Anemia,Henoch-Schonlein Purpura, Idiopathic Adrenal Atrophy, IdiopathicPulmonary Fibritis, IgA Nephropathy, Inflammatory Bowel Diseases,Insulin-dependent Diabetes Mellitus, Juvenile Arthritis, JuvenileDiabetes Mellitus (Type I), Lambert-Eaton Syndrome, Laminitis, LichenPlanus, Lupoid Hepatitis, Lupus, Lymphopenia, Meniere's Disease, MixedConnective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis,Pernicious Anemia, Polyglandular Syndromes, Presenile Dementia, PrimaryAgammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, PsoriaticArthritis, Raynauds Phenomenon, Recurrent Abortion, Reiter's Syndrome,Rheumatic Fever, Rheumatoid Arthritis, Sampter's Syndrome,Schistosomiasis, Schmidt's Syndrome, Scleroderma, Shulman's Syndrome,Sjorgen's Syndrome, Stiff-Man Syndrome, Sympathetic Ophthalmia, SystemicLupus Erythematosis, Takayasu's Arteritis, Temporal Arteritis,Thyroiditis, Thrombocytopenia, Thyrotoxicosis, Toxic EpidermalNecrolysis, Type B Insulin Resistance, Type I Diabetes Mellitus,Ulcerative Colitis, Uveitis, Vitiligo, Waldenstrom's Macroglobulemia,Wegener's Granulomatosis.

Multi-Medicant Therapy of Autoimmune Diseases. Methods for treating anautoimmune disease are also disclosed including administering to apatient in need thereof an effective amount of an Affinity MedicantLinker conjugates or Medicant Linker compound and another therapeuticagent known for the treatment of an autoimmune disease.

Treatment of Infectious Diseases. The Affinity Medicant Linkerconjugates and Medicant Linker compounds are useful for killing orinhibiting the multiplication of a cell that produces an infectiousdisease or for treating an infectious disease. The Affinity MedicantLinker conjugates and Medicant Linker compounds can be used accordinglyin a variety of settings for the treatment of an infectious disease in apatient. The Affinity Medicant Linker conjugates can be used to delivera Medicant unit to a target cell. In an embodiment of the presentinvention, AM binds to the infectious disease cell.

In various embodiments of the present invention, the AML or AMconjugates kill or inhibit the multiplication of cells that produce aparticular infectious disease including, but not limited to, Diphtheria,Pertussis, Occult Bacteremia, Urinary Tract Infection, Gastroenteritis,Cellulitis, Epiglottitis, Tracheitis, Adenoid Hypertrophy,Retropharyngeal Abcess, Impetigo, Ecthyma, Pneumonia, Endocarditis,Septic Arthritis, Pneumococca, Peritonitis, Bactermia, Meningitis, AcutePurulent Meningitis, Urethritis, Cervicitis, Proctitis, Pharyngitis,Salpingitis, Epididymitis, Gonorrhea, Syphilis, Listeriosis, Anthrax,Nocardiosis, Salmonella, Typhoid Fever, Dysentery, Conjunctivitis,Sinusitis, Brucellosis, Tullaremia, Cholera, Bubonic Plague, Tetanus,Necrotizing Enteritis, Actinomycosis, Mixed Anaerobic Infections,Syphilis, Relapsing Fever, Leptospirosis, Lyme Disease, Rat Bite Fever,Tuberculosis, Lymphadenitis, Leprosy, Chlamydia, Chlamydial Pneumonia,Trachoma, Inclusion Conjunctivitis Systemic Fungal Diseases:Histoplamosis, Coccidiodomycosis, Blastomycosis, Sporotrichosis,Cryptococcsis, Systemic Candidiasis, Aspergillosis, Mucormycosis,Mycetoma, Chromomycosis Rickettsial Diseases: Typhus, Rocky MountainSpotted Fever, Ehrlichiosis, Eastern Tick-Borne Rickettsioses,Rickettsialpox, Q Fever, Bartonellosis Parasitic Diseases: Malaria,Babesiosis, African Sleeping Sickness, Chagas' Disease, Leishmaniasis,Dum-Dum Fever, Toxoplasmosis, Meningoencephalitis, Keratitis,Entamebiasis, Giardiasis, Cryptosporidiasis, Isosporiasis,Cyclosporiasis, Microsporidiosis, Ascariasis, Whipworm Infection,Hookworm Infection, Threadworm Infection, Ocular Larva Migrans,Trichinosis, Guinea Worm Disease, Lymphatic Filariasis, Loiasis, RiverBlindness, Canine Heartworm Infection, Schistosomiasis, Swimmer's Itch,Oriental Lung Fluke, Oriental Liver Fluke, Fascioliasis,Fasciolopsiasis, Opisthorchiasis, Tapeworm Infections, Hydatid Disease,Alveolar Hydatid Disease Viral Diseases: Measles, Subacute sclerosingpanencephalitis, Common Cold, Mumps, Rubella, Roseola, Fifth Disease,Chickenpox, Respiratory syncytial virus infection, Croup, Bronchiolitis,Infectious Mononucleosis, Poliomyelitis, Herpangina, Hand-Foot-and-MouthDisease, Bornholm Disease, Genital Herpes, Genital Warts, AsepticMeningitis, Myocarditis, Pericarditis, Gastroenteritis, AcquiredImmunodeficiency Syndrome (AIDS), Human Immunodeficiency Virus (HIV),Reye's Syndrome, Kawasaki Syndrome, Influenza, Bronchitis, Viral“Walking” Pneumonia, Acute Febrile Respiratory Disease, Acutepharyngoconjunctival fever, Epidemic keratoconjunctivitis, HerpesSimplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2), Shingles,Cytomegalic Inclusion Disease, Rabies, Progressive MultifocalLeukoencephalopathy, Kuru, Fatal Familial Insomnia, Creutzfeldt-JakobDisease, Gerstmann-Straussler-Scheinker Disease, Tropical SpasticParaparesis, Western Equine Encephalitis, California Encephalitis, St.Louis Encephalitis, Yellow Fever, Dengue, Lymphocytic choriomeningitis,Lassa Fever, Hemorrhagic Fever, Hantvirus Pulmonary Syndrome, MarburgVirus Infections, Ebola Virus Infections, Smallpox.

Synthesis of AMCs with SMCC and Sulfo-SMCC linkers. In an embodiment ofthe present invention, an affinity medicant conjugate (AMC) 1000 isformed between an AM 1100 and a medicant 1350 by reacting the medicant1350 with succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate(SMCC) which forms an active intermediate that reacts with a sulfhydrylgroups on the AM 1100. In an embodiment of the present invention, theresulting AMC includes one or more molecules of the medicant 1350 boundto the AM 1100. In an embodiment of the present invention, the resultingAMC is not cleaved in the cytosol, but internalized and the AM 1100degraded by proteases in the cytosol until the medicant 1350 isreleased.

In an alternative embodiment of the present invention, an AMC 1000 isformed between an AM 1100 and a medicant 1350 by reacting the medicant1350 withsulfosuccinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene(Sulfo-SMCC) which forms an active intermediate that reacts with asulfhydryl groups on the AM 1100 to form a more water soluble AMC. In anembodiment of the present invention, the resulting AMC includes one ormore molecules of the medicant 1350 bound to the AM 1100. In anembodiment of the present invention, the resulting AMC is not cleaved inthe cytosol, but internalized and the AM 1100 degraded by proteases inthe cytosol until the medicant 1350 is released.

In an embodiment of the present invention, an AMC 1000 comprises an AM1100 bound to a medicant 1350 through an optional linker as illustratedin FIG. 1. In an embodiment of the present invention, an antibody 1110is bound to a linker 1200 which is bound to the medicant 1350. In anunexpected result, an AMC 1000 can retain both the receptor bindingactivity of the AM 1100 and the intracellular cytoactivity of themedicant 1350 in a single compound. In an embodiment of the presentinvention, an antibody 1110 is bound to a linker 1200 which is bound tothe medicant 1350. In an unexpected result, an antibody medicantconjugate can retain both the receptor binding activity of the antibody1110 and the intracellular cytoactivity of an acylfulvene in a singlecompound. Surprisingly, the antibody is capable of binding to apolypeptide receptor on cell populations thereby bringing theacylfulvene in contact with the cell population.

In an embodiment of the present invention, the medicant moiety is anacylfulvene moiety. An acylfulvene moiety includes irofulven derivatives(see structures shown in FIG. 2A, FIG. 2C, FIG. 2F, FIG. 2H, FIG. 2I,FIG. 2L, FIG. 2M, FIG. 2P, FIG. 2S and FIG. 2U) and illudin derivatives(see structures shown in FIG. 2B, FIG. 2D, FIG. 2E, FIG. 2G, FIG. 2J,FIG. 2K, FIG. 2N, FIG. 2O, FIG. 2Q, FIG. 2R, FIG. 2T, and FIG. 2V).

Amine Derivative. In an embodiment of the present invention, theirofulvene structures shown in FIG. 2A, FIG. 2C, FIG. 2F, FIG. 2H, FIG.2I, FIG. 2L and FIG. 2M and illudin structures shown in FIG. 2B, FIG.2D, FIG. 2E, FIG. 2G, FIG. 2J, FIG. 2K, FIG. 2N and FIG. 2O, where R₁denotes independently a carbon or a heteroatom containing nitrogen (N),oxygen (O) or sulphur (S); where R₆ denotes including —H, —CN, —CF₃, —O,—NH₂, —SO₃, —COOH—, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl in whichincorporated heteroatoms can be halogens (F, Cl, Br, I); nitrogen (N)functional groups including primary amines (—NH₂), secondary amines(—NH—), tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—),(—C(═N)R_(A)—), Azo (—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide(—C(═O)NR_(A)R_(B)) or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S)functional groups including thioethers (—S—), thiones (—C(═S)—,sulfoxides (—S(═O)—, sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))—or (—S(═O)(═NH)—, sulfhydryls (—SH), thiocyanate (—S—C(═N)—,isothiocyanate (—N═C(═S); oxygen (O) functional groups includinghydroxyl (—OH), carbonyl (—C(═O)—), aldehyde (—C(═O)H, carboxylate(COOH), ethers (—O—), esters (—OC(═O)—), carbonate (—O(C═O)O—); and R₂,R₃, R₄, R₅ denote either H, CH₃, or CH₂OH and where R₆ is NH₂ (an aminogroup) for an irofulvene derivative shown in FIG. 2A, FIG. 2C, FIG. 2F,FIG. 2H, FIG. 2I, FIG. 2L and FIG. 2M and illudin derivative shown inFIG. 2B, FIG. 2D, FIG. 2E, FIG. 2G, FIG. 2J, FIG. 2K, FIG. 2N and FIG.2O.

Table IA shows acylfulvene amine analogs which can be attached to abi-functional linker which can then be attached to a sulfhydryl reactinggroup of the AM using the reagent. In an embodiment of the presentinvention, the acylfulvene amino derivative shown in the first column ofTable IA is linked to the AM through the free sulfhydryl group of the AMusing the reagent identified in the second column of Table IA to formthe AMC.

FIG. 8A shows the structure of the analog 211 attached via the aminogroup using the SMPT linking reagents. FIG. 8B shows the structure ofthe analog 211 attached via the amino group using the SMCC linkingreagent. FIG. 8C shows the structure of the analog 211 attached via theamino group using the SIAB linking reagent.

Table IB shows acylfulvene amine analogs which can be attached to abi-functional linker which can then be attached to the AM via aphotoactivatable group at the other terminus using the reagent. In anembodiment of the present invention, the acylfulvene amino derivativeshown in the first column of Table IB is linked to the AM to the AMthrough the photoactivatable group at the other terminus using thereagent identified in the second column of Table IB to form the AMC.

Table IC shows acylfulvene amine analogs which can be attached to abi-functional linker which can then be attached to the AM through areactive amine group at the other terminus using the reagent. In anembodiment of the present invention, the acylfulvene amino derivativeshown in the first column of Table IC is linked to the AM through anamine reactive group using the reagent identified in the second columnof Table IC to form the AMC.

Table ID shows acylfulvene amine analogs which can be attached to abi-functional linker which can then be attached to the AM through analdehyde, carbonyl or carboxylate group at the other terminus using thereagent. In an embodiment of the present invention, the acylfulveneamino derivative shown in the first column of Table ID is linked to theAM through an aldehyde, carbonyl or carboxylate group at the otherterminus using the reagent identified in the second column of Table IDto form AMC.

Carboxyl Derivative. In an embodiment of the present invention, theirofulvene structures shown in FIG. 2A, FIG. 2C, FIG. 2F, FIG. 2H, FIG.2I, FIG. 2L and FIG. 2M and illudin structures shown in FIG. 2B, FIG.2D, FIG. 2E, FIG. 2G, FIG. 2J, FIG. 2K, FIG. 2N and FIG. 2O, where R₁denotes substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl in which incorporatedheteroatoms can be halogens (F, Cl, Br, I); nitrogen (N) functionalgroups including primary amines (—NH₂), secondary amines (—NH—),tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—), (—C(═N)R_(A)—), Azo(—N═N—), Cyanate isocyanate (—N═(C═O), amide (—C(═O)NR_(A)R_(B)) or(—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S) functional groups includingthioethers (—Sc—), thiones (—C(═S)—, sulfoxides (—S(═O)—, sulfones(—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))— or (—S(═O)(═NH)—, sulfhydryls(—SH), thiocyanate (—S—C(═N)—, isothiocyanate (—N═C(═S); oxygen (O)functional groups including hydroxyl (—OH), carbonyl (—C(═O)—), aldehyde(—C(═O)H, carboxylate (COOH), ethers (—O—), esters (—OC(═O)—), carbonate(—O(C═O)O—); and R₂, R₃, R₄, R₅ denote either H, CH₃, or CH₂OH and whereR₆ is CO₂H (a carboxyl group) for an irofulvene derivative shown in FIG.2A, FIG. 2C, FIG. 2F, FIG. 2H, FIG. 2I, FIG. 2L and FIG. 2M and illudinderivative shown in FIG. 2B, FIG. 2D, FIG. 2E, FIG. 2G, FIG. 2J, FIG.2K, FIG. 2N and FIG. 2O. R₅ is glycine or either an L or D amino acidincluding alanine, serine, threonine, cysteine, valine, leucine,isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan,aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine,arginine, alpha-methyl glycine or 2-dimethylglycine. R₅ can alsocomprise nonstandard amino acids to reduce nonspecific esterase activitypresent in blood and cells including homocysteine, selenocysteine,pyrrolysine, carnitine, hypusine, lanthionine, 2-aminoisobutyric acid,dehydroalanine, gamma-aminobutyric acid, ornithine, citrulline,α-Amino-n-butyric acid, Norvaline, Norleucine, Pipecolic acid,Alloisoleucine, α,β-diaminopropionic acid, α,γ-diaminobutyric acid,Allothreonine, α-Amino-n-heptanoic acid, Homoserine, β-Amino-n-butyricacid, β-Aminoisobutyric acid, α-Aminobutyric acid, isovaline, Sarcosine,N-ethyl glycine, N-propyl glycine, N-isopropyl glycine, N-methylalanine, N-ethyl alanine, N-methyl β-alanine, N-ethyl β-alanine,Isoserine, α-hydroxy-γ-aminobutyric acid, diaminopimelic acid,cystathione, aminoisobutyric acid, dehydroalanine, delta-aminolevulinicacid, 4-aminobenzoic acid, Hydroxyproline, Formylmethioinine,lanthionine, djenkolic acid, Pyroglutamic acid, Hypusine,carboxyglutamic acid, penicillamine, thialysine, quisqualic acid,canavine, azetidine-2-carboxylic acid. FIG. 9A shows the structure ofthe analog 038 attached via the carboxyl group using the EDC linkingreagent. FIG. 9B shows the structure of the analog 038 attached via thecarboxyl group using the CMC linking reagent. FIG. 9C shows structure ofthe analog 038 attached via the carboxyl group using DCC linkingreagent.

Table HA shows acylfulvene carboxylate analogs which can be attached toa bi-functional linker which can then be attached to a sulfhydrylreacting group of the AM. In an embodiment of the present invention, theacylfulvene carboxylate derivative shown in the first column of Table HAis linked to the AM through the free sulfhydryl group of the AM usingthe reagent identified in the second column of Table HA to form the AMC.

Table IIB shows acylfulvene carboxylate analogs which can be attached toa bi-functional linker, where the linker also contains aphotoactivatable reactive group which can attach to the AM. In anembodiment of the present invention, the acylfulvene carboxylatederivative shown in the first column of Table IIB is linked to the AMthrough the photoactivatable reactive group using the reagent identifiedin the second column of Table IIB to form the AMC.

Table IIC shows acylfulvene carboxylate analogs which can be attached toa bi-functional linker, where the linker also contains an amino reactivegroup which can attach to the AM. In an embodiment of the presentinvention, the acylfulvene carboxylate derivative shown in the firstcolumn of Table IIC is linked to the AM through the amino group usingthe reagent identified in the second column of Table IIC to form theAMC.

Azlactone Derivative. FIG. 10A shows the structure of the analog 038attached via the carboxyl group using DCC or DIC linking reagents in thepresence of glycine. FIG. 10B shows the structure of the analog 038attached via the carboxyl group using DCC or DIC linking reagents in thepresence of alanine. FIG. 10C shows the structure of the analog 106attached via the carboxyl group using DCC or DIC linking reagents in thepresence of valine.

Table HD shows acylfulvene carboxylate analogs which can be reacted toform acylfulvene azlactone derivatives where the azlactone reactivegroup can be used to attach to the AM. In an embodiment of the presentinvention, the acylfulvene derivative shown in the first column of TableHD is converted to the acylfulvene azlactone derivative (see FIG. 2P)using the reagent identified in the second column of Table HD to formthe AMC.

Carbonyl Derivative. In an embodiment of the present invention, theirofulvene structures shown in FIG. 2A, FIG. 2C, FIG. 2F, FIG. 2H, FIG.2I, FIG. 2L and FIG. 2M and illudin structures shown in FIG. 2B, FIG.2D, FIG. 2E, FIG. 2G, FIG. 2J, FIG. 2K, FIG. 2N and FIG. 2O, where R₁and R₇ denote independently substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl inwhich incorporated heteroatoms can be halogens (F, Cl, Br, I); nitrogen(N) functional groups including primary amines (—NH₂), secondary amines(—NH—), tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—),(—C(═N)R_(A)—), Azo (—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide(—C(═O)NR_(A)R_(B)) or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S)functional groups including thioethers (—S—), thiones (—C(═S)—,sulfoxides (—S(═O)—, sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))—or (—S(═O)(═NH)—, sulfhydryls (—SH), thiocyanate (—S—C(═N)—,isothiocyanate (—N═C(═S); oxygen (O) functional groups includinghydroxyl (—OH), carbonyl (—C(═O)—), aldehyde (—C(═O)H, carboxylate(COOH), ethers (—O—), esters (—OC(═O)—), carbonate (—O(C═O)O—); and R₂,R₃, R₄, R₅ denote either H, CH₃, or CH₂OH and where R₆ is CO—R₇ (acarbonyl linking group) for an irofulvene derivative shown in FIG. 2A,FIG. 2C, FIG. 2F, FIG. 2H, FIG. 2I, FIG. 2L and FIG. 2M and illudinderivative shown in FIG. 2B, FIG. 2D, FIG. 2E, FIG. 2G, FIG. 2J, FIG.2K, FIG. 2N and FIG. 2O.

FIG. 11A shows the structure of the analog 124 attached via the carbonylgroup using the AMBH linking reagent. FIG. 11B shows the structure ofthe analog 124 attached via the carbonyl group using the ABH linkingreagent. FIG. 11C shows the structure of the analog 201 attached via theM₂C₂H linking reagent.

Table IIIA shows acylfulvene carbonyl analogs which can be attached to abi-functional linker which can be attached to a sulfhydryl reactinggroup of the AM using the reagent. In an embodiment of the presentinvention, the acylfulvene carbonyl derivative shown in the first columnof Table IIIA is linked to the AM through the free sulfhydryl group ofthe AM using the reagent identified in the second column of Table IIIAto form the AMC.

Table IIIB shows acylfulvene carbonyl analogs which can be attached to abi-functional linker, where the linker also contains a photoactivatablereactive group which can attach to the AM using the reagent. In anembodiment of the present invention, the acylfulvene carbonyl derivativeshown in the first column of Table MB is linked to the AM through thephotoactivatable reactive group using the reagent identified in thesecond column of Table IIIB to form the AMC.

Table IIIC shows acylfulvene carbonyl analogs which can be attached to abi-functional linker, where the linker also contains an amine reactivegroup which can attach to the AM using the reagent. In an embodiment ofthe present invention, the acylfulvene carbonyl derivative shown in thefirst column of Table IIIC is linked to the AM through the amino groupusing the reagent identified in the second column of Table IIIC to formthe AMC.

Aldehyde Derivative. In an embodiment of the present invention, theirofulvene structures shown in FIG. 2A, FIG. 2C, FIG. 2F, FIG. 2H, FIG.2I, FIG. 2L and FIG. 2M and illudin structures shown in FIG. 2B, FIG.2D, FIG. 2E, FIG. 2G, FIG. 2J, FIG. 2K, FIG. 2N and FIG. 2O, where R₁denotes substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl in which incorporatedheteroatoms can be halogens (F, Cl, Br, I); nitrogen (N) functionalgroups including primary amines (—NH₂), secondary amines (—NH—),tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—), (—C(═N)R_(A)—), Azo(—N═N—), Cyanate isocyanate (—N═(C═O), amide (—C(═O)NR_(A)R_(B)) or(—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S) functional groups includingthioethers (—S—), thiones (—C(═S)—, sulfoxides (—S(═O)—, sulfones(—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))— or (—S(═O)(═NH)—, sulfhydryls(—SH), thiocyanate (—S—C(═N)—, isothiocyanate (—N═C(═S); oxygen (O)functional groups including hydroxyl (—OH), carbonyl (—C(═O)—), aldehyde(—C(═O)H, carboxylate (COOH), ethers (—O—), esters (—OC(═O)—), carbonate(—O(C═O)O—; and R₂, R₃, R₄, R₅ denote either H, CH₃, or CH₂OH and whereR₆ is HCO (an aldehyde group) for an irofulvene derivative shown in FIG.2A, FIG. 2C, FIG. 2F, FIG. 2H, FIG. 2I, FIG. 2L and FIG. 2M and illudinderivative shown in FIG. 2B, FIG. 2D, FIG. 2E, FIG. 2G, FIG. 2J, FIG.2K, FIG. 2N and FIG. 2O.

FIG. 12A shows the structure of the analog 010 attached via the aldehydegroup using the PDPH linking reagent. FIG. 12B shows the structure ofthe analog 010 attached via the aldehyde group using the ABH linkingreagent. FIG. 12C shows the structure of the analog 011 attached viaMPBH linking reagent.

Table IVA shows acylfulvene aldehyde analogs which can be attached to abi-functional linker which can be attached to a sulfhydryl reactinggroup of the AM using the reagent. In an embodiment of the presentinvention, the acylfulvene aldehyde derivative shown in the first columnof Table IVA is linked to the AM through the free sulfhydryl group ofthe AM using the reagent identified in the second column of Table IVA toform the AMC.

Table IVB shows acylfulvene aldehyde analogs which can be attached to abi-functional linker, where the linker also contains a photoactivatablereactive group which can attach to the AM using the reagent. In anembodiment of the present invention, the acylfulvene aldehyde derivativeshown in the first column of Table IVB is linked to the AM through thephotoactivatable reactive group using the reagent identified in thesecond column of Table IVB to form the AMC.

Table IVC shows acylfulvene aldehyde analogs which can be attached to abi-functional linker, where the linker also contains an amine reactivegroup which can attach to the AM using the reagent. In an embodiment ofthe present invention, the acylfulvene aldehyde derivative shown in thefirst column of Table IVC is linked to the AM through the amino groupusing the reagent identified in the second column of Table IVC to formthe AMC.

Alcohol Derivative. In an embodiment of the present invention, thestructures shown in FIG. 2C, FIG. 2D, and FIG. 2E, R₁ denotessubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl in which incorporatedheteroatoms can be halogens (F, Cl, Br, I); nitrogen (N) functionalgroups including primary amines (—NH₂), secondary amines (—NH—),tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—), (—C(═N)R_(A)—), Azo(—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide (—C(═O)NR_(A)R_(B))or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S) functional groupsincluding thioethers (—S—), thiones (—C(═S)—, sulfoxides (—S(═O)—,sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))— or (—S(═O)(═NH)—,sulfhydryls (—SH), thiocyanate (—S—C(═N)—, isothiocyanate (—N═C(═S);oxygen (O) functional groups including hydroxyl (—OH), carbonyl(—C(═O)—), aldehyde (—C(═O)H, carboxylate (COOH), ethers (—O—), esters(—OC(═O)—), carbonate (—O(C═O)O—; and R₂, R₃, R₄, R₅ denote either H,CH₃, or CH₂OH for an irofulven derivative (FIG. 2C), an illudin ringderivative (FIG. 2D) or an illudin alkyl derivative (FIG. 2E). FIG. 13Ashows the structure of the analog 009 attached via the alcohol groupusing the CDI linking reagent. FIG. 13B shows the structure of theanalog 009 attached via the alcohol group using the HSC linking reagent.FIG. 13C shows the structure of the medicant moiety Illudin M attachedvia the DSC linking reagent.

Table VA shows acylfulvene alcohol analogs which can be attached to abi-functional linker which can be attached to a sulfhydryl reactinggroup of the AM using the reagent. In an embodiment of the presentinvention, the acylfulvene alcohol derivative shown in the first columnof Table VA is linked to the AM through the free sulfhydryl group of theAM using the reagent identified in the second column of Table VA to formthe AMC.

Table VB shows acylfulvene alcohol analogs which can be attached to abi-functional linker, where the linker also contains an amine reactivegroup which can attach to the AM using the reagent. In an embodiment ofthe present invention, the acylfulvene alcohol derivative shown in thefirst column of Table VB is linked to the AM through the amino groupusing the reagent identified in the second column of Table VB to formthe AMC.

Sulfhydryl Derivative. In an embodiment of the present invention, thestructures shown in FIG. 2C, FIG. 2D, and FIG. 2E, R₁ and R₆ denoteindependently substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl in whichincorporated heteroatoms can be halogens (F, Cl, Br, I); nitrogen (N)functional groups including primary amines (—NH₂), secondary amines(—NH—), tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—),(—C(═N)R_(A)—), Azo (—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide(—C(═O)NR_(A)R_(B)) or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S)functional groups including thioethers (—S—), thiones (—C(═S)—,sulfoxides (—S(═O)—, sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))—or (—S(═O)(═NH)—, sulfhydryls (—SH), thiocyanate (—S—C(═N)—,isothiocyanate (—N═C(═S); oxygen (O) functional groups includinghydroxyl (—OH), carbonyl (—C(═O)—), aldehyde (—C(═O)H, carboxylate(COOH), ethers (—O—), esters (—OC(═O)—), carbonate (—O(C═O)O—; and R₂,R₃, R₄, R₅ denote either H, CH₃, or CH₂OH, and R₂, R₃, R₄, R₅ denoteeither H, CH₃, or CH₂OH and R₇ is SH or SS—R₈ for an irofulvenderivative (FIG. 2C), an illudin ring derivative (FIG. 2D) or an illudinalkyl derivative (FIG. 2E). FIG. 14A shows the structure of the analog051 attached via the sulfhydryl group using SMCC linking reagent. FIG.14B shows the structure of the analog 051 attached via the sulfhydrylgroup using MPBH linking reagent. FIG. 14C shows structure of the analog051 attached via sulfhydryl group using PDPH linking reagent.

In an embodiment of the present invention, analog 051 can be attached toan AM by attaching a disulfide bridge at 6′ position, a terminalcysteine or n-acetylcysteine group. Analog 051 has a free sulfhydrylgroup which can react with other sulfhydryl groups to produce adisulfide bond or alternatively react with specific sulfhydryl-reactinggroups such as malonic acid derivatives. The other sulfhydryl groups canbe on a linker, where the free sulfhydryl group will react withsulfhydryl reactive groups on the linkers, e.g., malonic acidderivatives such as SMCC. Alternatively a medicant with a freesulfhydryl can directly react with free sulfhydryl groups on an AM (suchas are present in cysteine residues).

Table VIA shows acylfulvene sulfhydryl analogs which can be attached toa bi-functional linker, where the linker also contains an amine reactivegroup which can attach to the AM using the reagent (a reducing agent canbe used to reduce the disulfide and generate a sulfhydryl group). In anembodiment of the present invention, the acylfulvene sulfhydrylderivative shown in the first column of Table VIA is linked to the AMthrough the free amino group of the bi-functional linker using thereagent identified in the second column of Table VIA to form the AMC.

Table VIB shows acylfulvene sulfhydryl analogs which can be attached toa bi-functional linker, where the linker also contains a sulfhydrylreacting group which can attach to the AM using the reagent (a reducingagent can be used to reduce the disulfide and generate a sulfhydrylgroup). In an embodiment of the present invention, the acylfulvenesulfhydryl derivative shown in the first column of Table VIB is linkedto the AM through the free sulfhydryl group of the bi-functional linkerusing the reagent identified in the second column of Table VIB to formthe AMC.

Table VIC shows acylfulvene sulfhydryl analogs which can be attached toa bi-functional linker, where the linker also contains aphotoactivatable reactive group which can attach to the AM using thereagent (a reducing agent can be used to reduce the disulfide andgenerate a sulfhydryl group). In an embodiment of the present invention,the acylfulvene sulfhydryl derivative shown in the first column of TableVIC is linked to the AM through the photoactivatable reactive group ofthe bi-functional linker using the reagent identified in the secondcolumn of Table VIC to form the AMC.

Table VID shows acylfulvene sulfhydryl analogs which can be attached toa bi-functional linker, where the linker also contains a carboxylatereactive group which can attach to the AM using the reagent (a reducingagent can be used to reduce the disulfide and generate a sulfhydrylgroup). In an embodiment of the present invention, the acylfulvenesulfhydryl derivative shown in the first column of Table VID is linkedto the AM through the carboxylate reactive group of the bi-functionallinker using the reagent identified in the second column of Table VID toform the AMC.

Halide Derivative. In an embodiment of the present invention, thestructures shown in FIG. 2C, FIG. 2D, and FIG. 2E, R₁ denotessubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl in which incorporatedheteroatoms can be halogens (F, Cl, Br, I); nitrogen (N) functionalgroups including primary amines (—NH₂), secondary amines (—NH—),tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—), (—C(═N)R_(A)—), Azo(—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide (—C(═O)NR_(A)R_(B))or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S) functional groupsincluding thioethers (—S—), thiones (—C(═S)—, sulfoxides (—S(═O)—,sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))— or (—S(═O)(═NH)—,sulfhydryls (—SH), thiocyanate (—S—C(═N)—, isothiocyanate (—N═C(═S);oxygen (O) functional groups including hydroxyl (—OH), carbonyl(—C(═O)—), aldehyde (—C(═O)H, carboxylate (COOH), ethers (—O—), esters(—OC(═O)—), carbonate (—O(C═O)O—; and R₂, R₃, R₄, R₅ denote either H,CH₃, or CH₂OH and X is a halogen for an irofulven derivative (FIG. 2C),an illudin ring derivative (FIG. 2D) or an illudin alkyl derivative(FIG. 2E).

In an embodiment of the present invention, the medicant moieties 4, 5,20, 53, 237 which contain halide groups can react in one of two ways.They will react directly with free sulfhydryl groups present onantibodies/proteins (e.g., on cysteine residues) or they can react withsulfhydryl groups on linkers (e.g., such as malonic acid derivativessuch as SMCC). FIG. 2I shows analog 20 linked to DSP (FIG. 21A), DTME(FIG. 21B) and SMPT (FIG. 21C).

Acyl azide or Azide Derivative. In an embodiment of the presentinvention, the structures shown in FIG. 2F and FIG. 2G, R₁ denotesindependently substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl in whichincorporated heteroatoms can be halogens (F, Cl, Br, I); nitrogen (N)functional groups including primary amines (—NH₂), secondary amines(—NH—), tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—),(—C(═N)R_(A)—), Azo (—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide(—C(═O)NR_(A)R_(B)) or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S)functional groups including thioethers (—S—), thiones (—C(═S)—,sulfoxides (—S(═O)—, sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))—or (—S(═O)(═NH)—, sulfhydryls (—SH), thiocyanate (—S—C(═N)—,isothiocyanate (—N═C(═S); oxygen (O) functional groups includinghydroxyl (—OH), carbonyl (—C(═O)—), aldehyde (—C(═O)H, carboxylate(COOH), ethers (—O—), esters (—OC(═O)—), carbonate (—O(C═O)O—; and R₂,R₃, R₄, R₅ denote either H, CH₃, OH, OCH₃, CH₂OH, CH₂CH₃, OCH₂CH₃ for anirofulven derivative (FIG. 2F) or an illudin derivative (FIG. 2G).

In an embodiment of the present invention, the medicant moieties 193,195, 299, 300, 307 can be photoactivated, with UV radiation. The acylazides and phenylazides do not need linkers, forming a reactive nitrenegroup that reacts with primary amines on proteins. The only caveat isthe reaction of the drug and protein must be carried out in the absenceof thiol reducing agents.

The azide must be on a ring system like a benzene or phenyl group, seeanalogs 193, 195, 300), 307 and 309.

Epoxide Derivative. In an embodiment of the present invention, thestructures shown in FIG. 2H, FIG. 2I, FIG. 2J and FIG. 2K, where R₁substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl in which incorporatedheteroatoms can be halogens (F, Cl, Br, I); nitrogen (N) functionalgroups including primary amines (—NH₂), secondary amines (—NH—),tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—), (—C(═N)R_(A)—), Azo(—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide (—C(═O)NR_(A)R_(B))or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S) functional groupsincluding thioethers (—S—), thiones (—C(═S)—, sulfoxides sulfones(—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))— or (—S(═O)(═NH)—, sulfhydryls(—SH), thiocyanate (—S—C(═N)—, isothiocyanate (—N═C(═S); oxygen (O)functional groups including hydroxyl (—OH), carbonyl (—C(═O)—), aldehyde(—C(═O)H, carboxylate (COOH), ethers (—O—), esters (—OC(═O)—), carbonate(—O(C═O)O—; and R₂, R₃, R₄, R₅ denote either H, CH₃, or CH₂OH, R₆denotes independently halogen, —CN, —CF₃, —OH, —NH₂, —SO₂, —COOH,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and R₂, R₃, R₄, R₅ denoteeither H, CH₃, OH, OCH₃, CH₂OH, CH₂CH₃, OCH₂CH₃ and X denotes aheteroatom including oxygen (O), sulfur (S), and nitrogen (N) forirofulven derivatives (FIG. 2H and FIG. 2I) or illudin derivatives (FIG.2J and FIG. 2K).

In an embodiment of the present invention, the medicant moieties 114epoxides react with carboxyl groups, thiols, amines and hydroxyl groups.For example, analog 114 can be linked to ABH, BMPA, or PDPH.

Example 1. Synthesis of Medicant 113. The Wittig reaction was performedon analog 10. First 65 mg CH₃PPh₃Br (0.185 mmol) in anhydrous THF wascooled to −75° C. and stirred for 1 hour. Then 200 μL of n—butyl lithium(0.183 mmol) was added very slowly to the flask while maintainingtemperature at −75° C., and a yellow precipitate formed. It was stirredfor another 1.5 hours then analog 10 (50 mg, 0.183 mmol) was slowlyadded while maintaining temperature at −75° C., followed by stirring for2.0 hours. The reaction was quenched with ammonium chloride, extractedwith CH₂Cl₂, washed with water, NaHCO₃, and saline. Dried over Na₂SO₄and concentrated. The residue was eluted through a column (10% ethylacetate in hexane) to give analog 113 as a solid.

Acroyl Derivative. In an embodiment of the present invention, thestructures shown in FIG. 2L, FIG. 2M, and FIG. 2N and FIGS. 2O, R₁ andR₆ denote independently substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl in whichincorporated heteroatoms can be halogens (F, Cl, Br, I); nitrogen (N)functional groups including primary amines (—NH₂), secondary amines(—NH—), tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—),(—C(═N)R_(A)—), Azo (—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide(—C(═O)NR_(A)R_(B)) or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S)functional groups including thioethers (—S—), thiones (—C(═S)—,sulfoxides (—S(═O)—, sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))—or (—S(═O)(═NH)—, sulfhydryls (—SH), thiocyanate (—S—C(═N)—,isothiocyanate (—N═C(═S); oxygen (O) functional groups includinghydroxyl (—OH), carbonyl (—C(═O)—), aldehyde (—C(═O)H, carboxylate(COOH), ethers (—O—), esters (—OC(═O)—), carbonate (—O(C═O)O—; and R₂,R₃, R₄, R₅ denote independently either H, CH₃, OH, OCH₃, CH₂OH, CH₂CH₃,OCH₂CH₃ and X denotes a heteroatom including oxygen (O), sulfur (S), andnitrogen (N) for irofulven derivatives (FIG. 2L and FIG. 2M) or illudinderivatives (FIG. 2N and FIG. 2O).

In an embodiment of the present invention, the medicant moieties 1300react predominately with sulfhydryl groups. Acroyl derivatives can reactin one of two ways. They will react directly with free sulfhydryl groupspresent on antibodies and proteins (e.g., on cysteine residues) or theywill react with sulthydryl groups on linkers (e.g., such as malonic acidderivatives such as SMCC).

Illudin1 linked to an Antibody. In various embodiments of the presentinvention, an AMC is made up of an antibody 1110 linked to an illudin1moiety 1301. Various embodiments of the invention, are directed to themethods for the preparation, use, and to pharmaceutical compositionscontaining an illudin1 moiety 1301 linked to an antibody 1110 to form anantibody medicant conjugate (AMC). In various embodiments the compoundsof the present invention, the AMC can have the general formula shown inFIG. 3A, where the antibody 1110 is bound to a linker 1200 which isbound to an illudin1 moiety 1301. In other various embodiments of thepresent invention, the compounds of the present AMC invention can havethe general formula shown in FIG. 3B, where a growth factor 1120 isbound to a linker 1200 which is bound to an illudin1 moiety 1301. Invarious embodiments the compounds of the present invention includestereoisomers, solvates, and pharmaceutically acceptable salts thereof,where the linker 1200 is as defined in Table X, and the illudin1 1301 isas defined below in Table XI.

Linker to bind Illudin to an Antibody. In an embodiment of the presentinvention, an antibody 1110 with a traditional linker 1240 to anilludin1 moiety 1301 binds to a receptor to which the antibody 1110 wasprepared and directs the illudin1 moiety 1301 to cell populationsexpressing the receptor. In an embodiment of the present invention, anantibody 1110 bound with a traditional linker 1240 to an illudin1 moiety1301 acts as an AM for a receptor and directs the illudin1 moiety 1301to tissues containing cells expressing the receptor. In an embodiment ofthe present invention, an antibody 1110 with a traditional linker 1240bound to an illudin1 moiety 1301 acts as an AM for a receptor anddirects the illudin1 moiety 1301 to tumors containing cells expressingthe receptor. In an embodiment of the present invention, an antibody1110 with a traditional linker 1240 bound to an illudin1 moiety 1301acts as an AM for a receptor and directs the illudin1 moiety 1301 totumors containing cells over-expressing the receptor compared to nontumor cells.

Linker to bind Illudin to Growth Factor. In an embodiment of the presentinvention, an illudin1 moiety 1301 linked via a traditional linker 1240to a growth factor 1120 binds to the growth factor receptor and directthe illudin1 moiety 1301 to cell populations expressing the receptor. Inan embodiment of the present invention, a growth factor 1120 linked viaa traditional linker 1240 to an illudin1 moiety 1301 acts as an AM forthe growth factor receptor and directs the illudin1 moiety 1301 totissues containing cells expressing the receptor. In an embodiment ofthe present invention, a growth factor 1120 linked via a traditionallinker 1240 to an illudin1 moiety 1301 acts as an AM for the growthfactor receptor and directs the illudin1 moiety 1301 to tumorscontaining cells expressing the receptor. In an embodiment of thepresent invention, a growth factor 1120 linked via a traditional linker1240 to an illudin1 moiety 1301 acts as an AM for the growth factorreceptor and directs the illudin1 moiety 1301 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

Linker to bind Illudin to Steroid. Whereby Illudin S, Illudin M, or oneof analogs 001 through 316 can be attached, either directly or with alinker, to a steroid which allows preferential binding to a celloverexpressing that particular receptor for that steroid and subsequentkilling of the cell (see e.g., Table VII).

Example 2. Synthesis of Medicant-Estrone 107. Analog 106 (see Example13) (139 mg 0.384 mmol, 1 equiv.), DMAP (4 mg, 0.03 mmol, 0.08 equiv.)and estrone (104.4 mg, 0.384 mmol, 1 equiv.) were dissolved in CH₂Cl₂(14 mL) at 0° C. To this solution was added CH₂Cl₂ solution of DCC (460μL, 1 M, 0.46 mmol, 1.2 equiv.) through a syringe. After 0.5 hours thesolution was raised to room temperature. After 2 hours the mixture wasfiltered and the filtrate was washed with dilute HCl (1.5%), saturatedNaHCO₃ and brine in sequence. The organic phase was then dried andevaporated. The residue was eluted through a column (CH₂Cl₂/Methanol,10:0.25) to give analog 107 (100 mg, 42%) as semisolid. Analog 107 canbe subsequently linked to estrone.

Example 3. Preparation of Medicant-Estradiol 108. Analog 038 (58.5 mg,0.2035 mmol), beta-estradiol (58.0 mg, 0.2150 mmol) and DMAP (5 mg.0.048 mmol) were dissolved in CH₂Cl₂ (5.6 mL) at 0° C. To this solutionwas added CH₂Cl₂ solution of DCC (250 μL, 1 M, 0.244 mmol), stirred for30 minutes, allowed to warm to room temperature then stirred for 1.5hours. The filtrate was washed with dilute HCl (1.5%), saturated NaHCO₃and brine in sequence. The organic phase was dried over Na₂SO₄, andevaporated. The residue was eluted through a column (100% CH₂Cl₂),fractions collected then eluted through a second column (CH₂Cl₂ plus0.5% methanol), to give analog 108 (45 mg) as a solid.

Table VII shows the cytotoxic data IC₅₀ values (micromolar, 2 hourexposure, N=3, mean±SD) for 108. MCF7 over express estrogenalpha-receptors. MCF7 cells are preferentially killed by 110 theacylfulvene-estrone analog and to a lesser extent 108 theacylfulvene-estradiol analog because estrone preferentially binds toalpha-receptor.

Example 4. Preparation of Medicant-Estradiol 109. Analog 106 (54.5 mg,0.15 mmol, 1 equiv.), β-estradiol (40.5 mg, 0.15 mmol), and DMAP (1.8mg, 0.015 mmol, 0.1 equiv.) were dissolved in CH₂Cl₂ (5 mL) at 0° C. Tothis solution was added CH₂Cl₂ solution of DCC (165 μL, 1 M, 0.165 mmol,1.1 equiv.). The mixture was raised to room temperature after 0.5 h.After another 2 h, the mixture was filtered. The filtrate was washedwith dilute HCl (1.5%), saturated NaHCO₃ and brine in sequence. Theorganic phase was dried and evaporated. The residue was eluted through acolumn (CH₂Cl₂/Methanol 10:0.25) to give analog 109 (55 mg, 60%) assemisolid.

Example 5. Preparation of Medicant-Estrone 110. Analog 038 (68 mg,0.2365 mmol), estrone (68.0 mg, 0.2160 mmol) and DMAP (5 mg. 0.048 mmol)were dissolved in CH₂Cl₂ (8.0 mL) at 0° C. To this solution was addedCH₂Cl₂ solution of DCC (300 μL, 1 M, 0.283 mmol), stirred for 30minutes, allowed to warm to room temperature then stirred for 0.5 hours.The filtrate was washed with dilute HCl (1.5%), saturated NaHCO₃ andbrine in sequence. The organic phase was dried over Na₂SO₄, andevaporated. The residue was eluted through a column (100% CH₂Cl₂),fractions collected then eluted through a second column (CH₂Cl₂ plus0.5% methanol), to give analog 110 (40 mg) as a solid.

Table VII shows the cytotoxic data IC₅₀ values (micromolar, 2 hourexposure, N=3, mean+SD) for 110. MCF7 cells over express estrogenalpha-receptors. MCF7 cells are preferentially killed by theacylfulvene-estrone analog 110 and to a lesser extent by theacylfulvene-estradiol analog 108 because estrone preferentially binds toalpha-receptor. In contrast, illudin M killed both ER negative and ERpositive cells to the same extent. The data in Table VII demonstratesthat compounds 108 and 110 are preferentially cytotoxic to cellsexpressing large numbers of estrogen receptors on their surface.

Example 6. Preparation of Medicant-Testosterone 111. Analog 038 (52.5mg, 0.182 mmol), testosterone (50.0 mg, 0.173 mmol) and DMAP (5 mg.0.048 mmol) were dissolved in CH₂Cl₂ (8.0 mL) at 0° C. To this solutionwas added CH₂Cl₂ solution of DCC (250 μL, 1 M), stirred for 30 minutes,allowed to warm to room temperature then stirred for 2 hours. Thefiltrate was washed with dilute HCl (1.5%), saturated NaHCO₃ and brinein sequence. The organic phase was dried over Na₂SO₄, and evaporated.The residue was eluted through a column (100% CH₂Cl₂ plus 0.5%methanol), to give analog 111 (15 mg) as a solid.

Example 7. Preparation of Medicant-Androsterone 112. Analog 038 (29 mg),androsterone (25.0 mg) and DMAP (5 mg. 0.048 mmol) were dissolved inCH₂Cl₂ (5.0 mL) at 0° C. To this solution was added CH₂Cl₂ solution ofDCC (150 μL, 1 M), stirred for 30 minutes, allowed to warm to roomtemperature then stirred for 2 hours. The filtrate was washed withdilute HCl (1.5%), saturated NaHCO₃ and brine in sequence. The organicphase was dried over Na₂SO₄, and evaporated. The residue was elutedthrough a column (2:3 ethyl acetate:hexane) to give analog 112 (15 mg)as a solid.

In an embodiment of the present invention, an illuclin2 moiety 1302linked via a traditional linker 1240 to a steroid 1140 bind to receptorsfor the steroid and directs the illuclin2 moiety 1302 to cellpopulations expressing the receptor. In an embodiment of the presentinvention, a steroid 1140 linked via a traditional linker 1240 to anilluclin2 moiety 1302 acts as an AM for the steroid hormone receptor anddirects the illuclin2 moiety 1302 to tissues containing cells expressingthe receptor. In an embodiment of the present invention, a steroid 1140linked via a traditional linker 1240 to an illudin2 moiety 1302 acts asan AM for the steroid hormone receptor and directs the illuclin2 moiety1302 to tumors containing cells over-expressing the receptor compared tonon tumor cells.

Example 4. Preparation of Medicant-Estradiol 109. Analog 106 (54.5 mg,0.15 mmol, 1 equiv.), β-estradiol (40.5 mg, 0.15 mmol), and DMAP (1.8mg, 0.015 mmol, 0.1 equiv.) were dissolved in CH₂Cl₂(5 mL) at 0° C. Tothis solution was added CH₂Cl₂ solution of DCC (165 μL, 1 M, 0.165 mmol,1.1 equiv.). The mixture was raised to room temperature after 0.5 h.After another 2 h, the mixture was filtered. The filtrate was washedwith dilute HCl (1.5%), saturated NaHCO₃ and brine in sequence. Theorganic phase was dried and evaporated. The residue was eluted through acolumn (CH₂Cl₂/Methanol 10:0.25) to give analog 109 (55 mg, 60%) assemisolid.

Example 5. Preparation of Medicant-Estrone 110. Analog 038 (68 mg,0.2365 mmol), estrone (68.0 mg, 0.2160 mmol) and DMAP (5 mg. 0.048 mmol)were dissolved in CH₂Cl₂ (8.0 mL) at 0° C. To this solution was addedCH₂Cl₂ solution of DCC (300 μL, 1 M, 0.283 mmol), stirred for 30minutes, allowed to warm to room temperature then stirred for 0.5 hours.The filtrate was washed with dilute HCl (1.5%), saturated NaHCO₃ andbrine in sequence. The organic phase was dried over Na₂SO₄, andevaporated. The residue was eluted through a column (100% CH₂Cl₂),fractions collected then eluted through a second column (CH₂Cl₂ plus0.5% methanol), to give analog 110 (40 mg) as a solid.

Table VII shows the cytotoxic data IC₅₀ values (micromolar, 2 hourexposure, N=3, mean+SD) for 110. MCF7 cells over express estrogenalpha-receptors. MCF7 cells are preferentially killed by theacylfulvene-estrone analog 110 and to a lesser extent by theacylfulvene-estradiol analog 108 because estrone preferentially binds toalpha-receptor. In contrast, illudin M killed both ER negative and ERpositive cells to the same extent. The data in Table VII demonstratesthat compounds 108 and 110 are preferentially cytotoxic to cellsexpressing large numbers of estrogen receptors on their surface.

Example 6. Preparation of Medicant-Testosterone 111. Analog 038 (52.5mg, 0.182 mmol), testosterone (50.0 mg, 0.173 mmol) and DMAP (5 mg.0.048 mmol) were dissolved in CH₂Cl₂ (8.0 mL) at 0° C. To this solutionwas added CH₂Cl₂ solution of DCC (250 μL, 1 M), stirred for 30 minutes,allowed to warm to room temperature then stirred for 2 hours. Thefiltrate was washed with dilute HCl (1.5%), saturated NaHCO₃ and brinein sequence. The organic phase was dried over Na₂SO₄, and evaporated.The residue was eluted through a column (100% CH₂Cl₂ plus 0.5%methanol), to give analog 111 (15 mg) as a solid.

Example 7. Preparation of Medicant-Androsterone 112. Analog 038 (29 mg),androsterone (25.0 mg) and DMAP (5 mg. 0.048 mmol) were dissolved inCH₂Cl₂ (5.0 mL) at 0° C. To this solution was added CH₂Cl₂ solution ofDCC (150 μL, 1 M), stirred for 30 minutes, allowed to warm to roomtemperature then stirred for 2 hours. The filtrate was washed withdilute HCl (1.5%), saturated NaHCO₃ and brine in sequence. The organicphase was dried over Na₂SO₄, and evaporated. The residue was elutedthrough a column (2:3 ethyl acetate:hexane) to give analog 112 (15 mg)as a solid.

In an embodiment of the present invention, an illudin2 moiety 1302linked via a traditional linker 1240 to a steroid 1140 bind to receptorsfor the steroid and directs the illudin2 moiety 1302 to cell populationsexpressing the receptor. In an embodiment of the present invention, asteroid 1140 linked via a traditional linker 1240 to an illudin2 moiety1302 acts as an AM for the steroid hormone receptor and directs theilludin2 moiety 1302 to tissues containing cells expressing thereceptor. In an embodiment of the present invention, a steroid 1140linked via a traditional linker 1240 to an illudin2 moiety 1302 acts asan AM for the steroid hormone receptor and directs the illudin2 moiety1302 to tumors containing cells over-expressing the receptor compared tonon tumor cells factor receptor and directs the illudin2 moiety 1302 totissues containing cells expressing the receptor. In an embodiment ofthe present invention, a growth factor 1120 linked via a FSB linker 1220to an illudin2 moiety 1302 acts as an AM for the growth factor receptorand directs the illudin2 moiety 1302 to tumors containing cellsexpressing the receptor. In an embodiment of the present invention, agrowth factor 1120 linked via a FSB linker 1220 to an illudin2 moiety1302 acts as an AM for the growth factor receptor and directs theilludin2 moiety 1302 to tumors containing cells over-expressing thereceptor compared to non tumor cells.

FSB Linker to bind Illudin to a Steroid. In an embodiment of the presentinvention, an illudin2 moiety 1302 linked via a FSB linker 1220 to asteroid 1140 binds to receptors for the steroid and direct the illudin2moiety 1302 to cell populations expressing the receptor. In anembodiment of the present invention, a steroid 1140 linked via a FSBlinker 1220 to an illudin2 moiety 1302 acts as a ligand for the steroidhormone receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a steroid 1140 linked via a FSB linker 1220 to anilludin2 moiety 1302 acts as a ligand for the steroid hormone receptorand directs the illudin2 moiety 1302 to tumors containing cellsexpressing the receptor. In an embodiment of the present invention, asteroid 1140 linked via a FSB linker 1220 to an illudin2 moiety 1302acts as an AM for the steroid hormone receptor and directs the illudin2moiety 1302 to tissues containing cells expressing the receptor. In anembodiment of the present invention, a steroid 1140 linked via a FSBlinker 1220 to an illudin2 moiety 1302 acts as an AM for the steroidhormone receptor and directs the illudin2 moiety 1302 to tumorscontaining cells expressing the receptor. In an embodiment of thepresent invention, a steroid 1140 linked via a FSB linker 1220 to anilludin2 moiety 1302 acts as an AM for the steroid hormone receptor anddirects the illudin2 moiety 1302 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

FSB Linker to bind Illudin to an Anti-angiogenic peptide. In anembodiment of the present invention, an illudin2 moiety 1302 linked viaa FSB linker 1220 to an anti-angiogenic peptide 1130 binds to receptorsfor the anti-angiogenic peptide and directs the illudin2 moiety 1302 tocell populations expressing the receptor. In an embodiment of thepresent invention, an anti-angiogenic peptide 1130 linked via a FSBlinker 1220 to an illudin2 moiety 1302 acts as a ligand for theanti-angiogenic peptide receptor and directs the illudin2 moiety 1302 totissues containing cells expressing the receptor. In an embodiment ofthe present invention, an anti-angiogenic peptide 1130 linked via a FSBlinker 1220 to an illudin2 moiety 1302 acts as a ligand for the peptidereceptor and directs the illudin2 moiety 1302 to tumors containing cellsexpressing the receptor. In an embodiment of the present invention, ananti-angiogenic peptide 1130 linked via a FSB linker 1220 to an illudin2moiety 1302 acts as an AM for the anti-angiogenic peptide receptor anddirects the illudin2 moiety 1302 to tissues containing cells expressingthe receptor. In an embodiment of the present invention, ananti-angiogenic peptide 1130 linked via a FSB linker 1220 to an illudin2moiety 1302 acts as an AM for the peptide receptor and directs theilludin2 moiety 1302 to tumors containing cells expressing the receptor.In an embodiment of the present invention, an anti-angiogenic peptide1130 linked via a FSB linker 1220 to an illudin2 moiety 1302 acts as anAM for the peptide receptor and directs the illudin2 moiety 1302 totumors containing cells over-expressing the receptor compared to nontumor cells.

FSB Linker to bind Illudin to an Integrin binding peptide. In anembodiment of the present invention, an illudin2 moiety 1302 linked viaa FSB linker 1220 to an integrin binding peptide 1150 binds to receptorsfor the integrin binding peptide and directs the illudin2 moiety 1302 tocell populations expressing the receptor. In an embodiment of thepresent invention, an integrin binding peptide 1150 linked via a FSBlinker 1220 to an illudin2 moiety 1302 acts as a ligand for the integrinbinding peptide receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, an integrin binding peptide 1150 linked FSB linker1220 to an illudin2 moiety 1302 acts as a ligand for the integrinbinding peptide receptor and directs the illudin2 moiety 1302 to tumorscontaining cells expressing the receptor. In an embodiment of thepresent invention, an integrin binding peptide 1150 linked via a FSBlinker 1220 to an illudin2 moiety 1302 acts as an AM for the integrinbinding peptide receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, an integrin binding peptide 1150 linked FSB linker1220 to an illudin2 moiety 1302 acts as an AM for the integrin bindingpeptide receptor and directs the illudin2 moiety 1302 to tumorscontaining cells expressing the receptor. In an embodiment of thepresent invention, an integrin binding peptide 1150 linked FSB linker1220 to an illudin2 moiety 1302 acts as an AM for the integrin bindingpeptide receptor and directs the illudin2 moiety 1302 to tumorscontaining cells over-expressing the receptor compared to non tumorcells.

FSB Linker to bind Illudin to a Pro-peptide. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via a FSB linker 1220to a pro-peptide 1160 is cleaved by an enzyme 1165 to generate thepeptide 1161 and thereafter binds to receptors for the peptide anddirects the illudin2 moiety 1302 to cell populations expressing thereceptor. In an embodiment of the present invention, a pro-peptide 1160linked via a FSB linker 1220 to an illudin2 moiety 1302 is cleaved by anenzyme 1165 and thereafter the peptide 1161 acts as a ligand for thepeptide receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a pro-peptide 1160 linked via a FSB linker 1220 to anilludin2 moiety 1302 is cleaved by an enzyme 1165 and thereafter thepeptide 1161 directs the illudin2 moiety 1302 to tumors containing cellsexpressing the receptor. In an embodiment of the present invention, apro-peptide 1160 linked via a FSB linker 1220 to an illudin2 moiety 1302is cleaved by an enzyme 1165 and thereafter the peptide 1161 acts as anAM for the peptide receptor and directs the illudin2 moiety 1302 totissues containing cells expressing the receptor. In an embodiment ofthe present invention, a pro-peptide 1160 linked via a FSB linker 1220to an illudin2 moiety 1302 is cleaved by an enzyme 1165 and thereafterthe peptide 1161 directs the illudin2 moiety 1302 to tumors containingcells expressing the receptor. In an embodiment of the presentinvention, a pro-peptide 1160 linked via a FSB linker 1220 to anilludin2 moiety 1302 is cleaved by an enzyme 1165 and thereafter thepeptide 1161 directs the illudin2 moiety 1302 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

FSB Linker to bind Illudin to a Glycopeptide. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via a FSB linker 1220to a glycopeptide 1170 with biological activity binds to receptors forthe glycopeptide 1170 and directs the illudin2 moiety 1302 to cellpopulations expressing the receptor. In an embodiment of the presentinvention, a glycopeptide 1170 linked via a FSB linker 1220 to anilludin2 moiety 1302 acts as a ligand for the glycopeptide receptor anddirects the illudin2 moiety 1302 to tissues containing cells expressingthe receptor. In an embodiment of the present invention, a glycopeptide1170 linked via a FSB linker 1220 to an illudin2 moiety 1302 acts as aligand for the glycopeptide receptor and directs the illudin2 moiety1302 to tumors containing cells expressing the receptor. In anembodiment of the present invention, a glycopeptide 1170 linked via aFSB linker 1220 to an illudin2 moiety 1302 acts as an AM for theglycopeptide receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a glycopeptide 1170 linked via a FSB linker 1220 toan illudin2 moiety 1302 acts as an AM for the glycopeptide receptor anddirects the illudin2 moiety 1302 to tumors containing cells expressingthe receptor. In an embodiment of the present invention, a glycopeptide1170 linked via a FSB linker 1220 to an illudin2 moiety 1302 acts as anAM for the glycopeptide receptor and directs the illudin2 moiety 1302 totumors containing cells over-expressing the receptor compared to nontumor cells.

FSB Linker to bind Illudin to a Lipid. In an embodiment of the presentinvention, an illudin2 moiety 1302 linked via a FSB linker 1220 to alipid 1180 with biological activity binds to receptors for the lipid1180 and directs the illudin2 moiety 1302 to cell populations expressingthe lipid. In an embodiment of the present invention, a lipid 1180linked via a FSB linker 1220 to an illudin2 moiety 1302 acts as a ligandfor the lipid receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a lipid 1180 linked via a FSB linker 1220 to anilludin2 moiety 1302 acts as a ligand for the lipid receptor and directsthe illudin2 moiety 1302 to tumors containing cells expressing thereceptor. In an embodiment of the present invention, a lipid 1180 linkedvia a FSB linker 1220 to an illudin2 moiety 1302 acts as an AM for thelipid receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a lipid 1180 linked via a FSB linker 1220 to anilludin2 moiety 1302 acts as an AM for the lipid receptor and directsthe illudin2 moiety 1302 to tumors containing cells over-expressing thereceptor compared to non tumor cells.

FSB Linker to bind Illudin to a Peptide. In an embodiment of the presentinvention, an illudin2 moiety 1302 linked via a FSB linker 1220 to apeptide 1190 with biological activity binds to the peptide receptor anddirects the illudin2 moiety 1302 to cell populations expressing thereceptor. In an embodiment of the present invention, a peptide 1190linked via a FSB linker 1220 to an illudin2 moiety 1302 acts as a ligandfor the peptide receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a peptide 1190 linked via a FSB linker 1220 to anilludin2 moiety 1302 acts as a ligand for the peptide receptor anddirects the illudin2 moiety 1302 to tumors containing cells expressingthe receptor. In an embodiment of the present invention, a peptide 1190linked via a FSB linker 1220 to an illudin2 moiety 1302 acts as an AMfor the peptide receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a peptide 1190 linked via a FSB linker 1220 to anilludin2 moiety 1302 acts as an AM for the peptide receptor and directsthe illudin2 moiety 1302 to tumors containing cells expressing thereceptor. In an embodiment of the present invention, a peptide 1190linked via a FSB linker 1220 to an illudin2 moiety 1302 acts as an AMfor the peptide receptor and directs the illudin2 moiety 1302 to tumorscontaining cells over-expressing the receptor compared to non tumorcells.

Linker to bind Illudin2 to a Glycopeptide. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via a traditionallinker 1240 to a glycopeptide 1170 with biological activity binds toreceptors for the glycopeptide 1170 and directs the illudin2 moiety 1302to cell populations expressing the receptor. In an embodiment of thepresent invention, a glycopeptide 1170 linked via a traditional linker1240 to an illudin2 moiety 1302 acts as a ligand for the glycopeptidereceptor and directs the illudin2 moiety 1302 to tissues containingcells expressing the receptor. In an embodiment of the presentinvention, a glycopeptide 1170 linked via a traditional linker 1240 toan illudin2 moiety 1302 acts as a ligand for the glycopeptide receptorand directs the illudin2 moiety 1302 to tumors containing cellsexpressing the receptor.

Linker to bind Illudin2 to a Lipid. In an embodiment of the presentinvention, an illudin2 moiety 1302 linked via a traditional linker 1240to a lipid 1180 with biological activity binds to receptors for thelipid 1180 and directs the illudin2 moiety 1302 to cell populationsexpressing the lipid receptor or lipid binding protein. In an embodimentof the present invention, a lipid 1180 linked via a traditional linker1240 to an illudin2 moiety 1302 acts as a ligand for the lipid receptorand directs the illudin2 moiety 1302 to tissues containing cellsexpressing the receptor. In an embodiment of the present invention, alipid 1180 linked via a traditional linker 1240 to an illudin2 moiety1302 acts as a ligand for the lipid receptor and directs the illudin2moiety 1302 to tumors containing cells expressing the receptor.

Linker to bind Illudin2 to a Growth Factor. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via a traditionallinker 1240 to a growth factor 1120 with biological activity binds toreceptors for the growth factor 1120 and directs the illudin2 moiety1302 to cell populations expressing the growth factor receptor. In anembodiment of the present invention, a growth factor 1120 linked via atraditional linker 1240 to an illudin2 moiety 1302 acts as a ligand forthe growth factor receptor and directs the illudin2 moiety 1302 totissues containing cells expressing the receptor. In an embodiment ofthe present invention, a growth factor 1120 linked via a traditionallinker 1240 to an illudin2 moiety 1302 acts as a ligand for the growthfactor receptor and directs the illudin2 moiety 1302 to tumorscontaining cells expressing the receptor.

Linker to bind Illudin2 to an anti-angiogenic peptide. In an embodimentof the present invention, an illudin2 moiety 1302 linked via atraditional linker 1240 to an anti-angiogenic peptide 1130 withbiological activity binds to receptors for the anti-angiogenic peptide1130 and directs the illudin2 moiety 1302 to cell populations expressingthe anti-angiogenic peptide receptor. In an embodiment of the presentinvention, an anti-angiogenic peptide 1130 linked via a traditionallinker 1240 to an illudin2 moiety 1302 acts as a ligand for theanti-angiogenic peptide receptor and directs the illudin2 moiety 1302 totissues containing cells expressing the receptor. In an embodiment ofthe present invention, an anti-angiogenic peptide 1130 linked via atraditional linker 1240 to an illudin2 moiety 1302 acts as a ligand forthe anti-angiogenic peptide receptor and directs the illudin2 moiety1302 to tumors containing cells expressing the receptor.

Linker to bind Illudin2 to a Steroid. In an embodiment of the presentinvention, an illudin2 moiety 1302 linked via a traditional linker 1240to a steroid 1140 with biological activity binds to receptors for thesteroid 1140 and directs the illudin2 moiety 1302 to cell populationsexpressing the steroid receptor. In an embodiment of the presentinvention, a steroid 1140 linked via a traditional linker 1240 to anilludin2 moiety 1302 acts as a ligand for the steroid receptor anddirects the illudin2 moiety 1302 to tissues containing cells expressingthe receptor. In an embodiment of the present invention, a steroid 1140linked via a traditional linker 1240 to an illudin2 moiety 1302 acts asa ligand for the steroid receptor and directs the illudin2 moiety 1302to tumors containing cells expressing the receptor.

Linker to bind Illudin2 to an Integrin binding protein. In an embodimentof the present invention, an illudin2 moiety 1302 linked via atraditional linker 1240 to an integrin binding protein 1150 withbiological activity binds to receptors for the integrin binding protein1150 and directs the illudin2 moiety 1302 to cell populations expressingthe integrin binding protein receptor. In an embodiment of the presentinvention, an integrin binding protein 1150 linked via a traditionallinker 1240 to an illudin2 moiety 1302 acts as a ligand for the integrinbinding protein receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, an integrin binding protein 1150 linked via atraditional linker 1240 to an illudin2 moiety 1302 acts as a ligand forthe integrin binding protein receptor and directs the illudin2 moiety1302 to tumors containing cells expressing the receptor.

Linker to bind Illudin2 to Folate. In an embodiment of the presentinvention, an illudin2 moiety 1302 linked via a traditional linker 1240to folate 1185 binds to receptors for the folate 1185 and directs theilludin2 moiety 1302 to cell populations expressing the folate receptor.In an embodiment of the present invention, folate 1185 linked via atraditional linker 1240 to an illudin2 moiety 1302 acts as a ligand forthe folate receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, folate 1185 linked via a traditional linker 1240 toan illudin2 moiety 1302 acts as a ligand for the folate receptor anddirects the illudin2 moiety 1302 to tumors containing cells expressingthe receptor.

Linker to bind Illudin2 to a Peptide. In an embodiment of the presentinvention, an illudin2 moiety 1302 linked via a traditional linker 1240to a peptide 1190 with biological activity binds to the peptide receptorand directs the illudin2 moiety 1302 to cell populations expressing thereceptor. In an embodiment of the present invention, a peptide 1190linked via a traditional linker 1240 to an illudin2 moiety 1302 acts asa ligand for the peptide receptor and directs the illudin2 moiety 1302to tissues containing cells expressing the receptor. In an embodiment ofthe present invention, a peptide 1190 linked via a traditional linker1240 to an illudin2 moiety 1302 acts as a ligand for the peptidereceptor and directs the illudin2 moiety 1302 to tumors containing cellsexpressing the receptor.

Mall linker 1211. Synthesis of mono-protected linkers. Maleimide orMaleic derivatives of acylfulvenes, Illudins and Syn-illudins can reactdirectly with thiol groups on antibodies, proteins or with primaryamines. When the Mall linker is used there is no need to attach theacylfulvene or illudin analog to linker as it is already incorporatedinto the analog (see FIG. 2S, FIG. 2T, FIG. 2U, and FIG. 2V). BothIlludin and Acylfulvene derivatives have been synthesized (e.g., analog189, analog 190, analog 217, and analog 218, see also FIG. 19), and datademonstrating their in vitro activity and selectivity towards cellsexpressing a specific surface antigen are shown in FIG. 19.

Example 12. In an embodiment of the present invention, AMC's 189, 190,217, 218 incorporating Mall linkers were synthesized. Unexpectedly, theAMC 189, 190, 217, 218 were found to be cytotoxic with nM activity (seeTable XV).

In an embodiment of the present invention, an AMC in which analog 218bound to an antibody using the Mall linker (FIG. 19) shows superioractivity compared to current antibodies medicinally used (e.g.Herceptin). Table XV shows the cytotoxicity data for AMC's 189, 190,217, 218 incorporating the Mall linker.

In an embodiment of the present invention, the Mall linker was attachedto the acylfulvene. In this manner, the medicant-linker will binddirectly to sulfhydryl groups on an AM, e.g., antibody or peptidescontaining a cysteine (with a sulfhydryl group). This novelmedicant-linker allows the generation of toxin-peptide conjugates thatcan be cleaved by enzymes. Alternatively toxin-peptide conjugates can beprepared that will bind directly to tumor associated antigens (PMSA),specific integrins, or anti-angiogenic peptides.

Synthesis of Linkers. The synthesis of medicant moieties bound tolinkers can be carried out using the following strategies: React R—NH₂with H—N═C═S to form isothiourea R—NH—C(═S)—NH₂. React R—NH₂ withH—N═C═O to form isourea R—NH—C(═O)—NH₂. React R—NH₂ with acyl azide toform RC(═O)NHR. React R—NH₂ with NHS ester to form RC(═O)NHR. Reactamine with sulfonyl chloride to form sulfonamide bond R(S(═O)(═O)NHR.React amine with imidoester to form amidine linkage RCH₂C(═NH₂)NHR.React amine with succinic acid to make amide bond with carboxylate ion.React imidoester with amine to form amidine bond RCH₂C(═NH₂+)NHR (seealso Table X).

Azlactone linker Reactions of carbodiimides such asdicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC) with acarboxylic acid yields a highly reactive O-acylisourea. Duringartificial protein synthesis (such as Fmoc solid-state synthesizers),the C-terminus is often used as the attachment site on which the aminoacid monomers are added. To enhance the electrophilicity of carboxylategroup, the negatively charged oxygen must first be “activated” into abetter leaving group and carbodiimides can be used for this purpose. Thenegatively charged oxygen will act as a nucleophile, attacking thecentral carbon in DCC. DCC is temporarily attached to the formercarboxylate group (which is now an ester group), making nucleophilicattack by an amino group (on the attaching amino acid) to the formerC-terminus (carbonyl group) more efficient.

When the Illudin, Syn-Illudin, or Acylfulvene carboxylic acid analog isactivated by DCC or DIC in the presence of an amino acid theDCC-activated carboxylate will react with the amino acid to form anazlactone (FIG. 2P, FIG. 2Q, FIG. 2R, and FIG. 10). This aminoacid-derived azlactone will react with primary amines, undergo ringopening, and forms an amide bond.

Example 13. Synthesis of Medicant 106. Illudin M (450 mg, 1.845 mmol, 1equiv.), glutaric anhydride (2.10 g, 18.45 mmol, 10 equiv.) and DMAP(171 mg, 1.4 mmol, 0.76 equiv.) were dissolved in CH₂Cl₂ (5 mL) at roomtemperature. After 3.5 hours the mixture was taken up by CH₂Cl₂, whichwas washed with water, and brine in sequence. It was then dried andevaporated. The residue was eluted through a column (Hexane/EtOAc 4:1)to give analog 106 (365 mg, 55%) as a liquid. UV (CHCl₃) λ nm (ε): 309(3387).

Analog 106 was generated from illudin M as outlined in Example 13. Thecarboxylic acid derivative was activated using DCC/DMAP to synthesizesteroid AFC's 107 and 109. In addition, Irofulven carboxylic acidderivative, analog 038 was activated using DCC/DMAP to produce analogs108, 110, 111, and 112. In general, carboxylate group containingcompounds can be activated using a carbodiimide in the presence of anamino acid to form an azlactone. The azlactone formed will reactspontaneously with primary amine groups on an amino acid, a peptide, anantibody, a protein, or another drug, and undergo ring opening with theformation of an amide bond. For proteins, antibodies and peptides theamino acids capable of reacting with the azlactone derivative includesarginine and lysine.

To form an Illudin derived azlactone active drug-linker moiety, eitheranalog 106 or analog 038 can be activated by DCC/DMAP in the presence ofa small amino acid such as glycine to form the azlactone. DCC cannot beadded without the presence of an amine containing target (such as theglycine) or the activated carboxylate reacts with another carboxylate toform a symmetrical anhydride. The azlactone formed will reactspontaneously with primary amine groups on a peptide, an antibody, aprotein, or a medicant.

Example 14. Activation of analog 038 by DCC to form medicant-azlactone.Part A: Production of Azlactone from carboxylate Acylfulvene analog:Analog 038 (58.5 mg, 0.2035 mmol), and DMAP (5 mg. 0.048 mmol) weredissolved in CH₂Cl₂ (5.6 mL) at 0° C. The desired amino acid (such asglycine) is added in an equimolar amount. Note that amino acids havingsubstitutions on the C4 carbon (such as alpha-methyl glycine or2-dimethylglycine) are preferred over conventional amino acids assubstitution cannot occur at the C4 position after ring-opening and allnucleophilic coupling reactions must occur at the C5 position, resultingonly in the desired amide-bond formation with the amine-containingmolecule. To this solution was added CH₂Cl₂ solution of DCC (250 μL, 1M, 0.244 mmol), stirred for 30 minutes, allowed to warm to roomtemperature then stirred for 1.5 hours. The filtrate was washed withdilute HCl (1.5%), saturated NaHCO₃ and brine in sequence. The organicphase was dried over Na₂SO₄, and evaporated. The residue was elutedthrough a column (100% CH₂Cl₂ plus 0.5% methanol), to give the desiredazlactone analog as a solid. Part B: Coupling of Azlactone to theprotein component (reacting with primary amines on amino acids such asthe one on lysine): The typical protein coupling reaction consists ofthe Azlactone suspended in buffer [25 mM sodium phosphate, 150 mM NaCl(pH 7.5)] and the desired amount of protein (20 μg to 5.0 mg) is added.The mixture is gently rocked for 60 minutes, then the reactionterminated by the addition of the blocking reagent, 1.0 ml of 1.0 Methanolamine in 25 mM sodium pyrophosphate (titrated to pH 9.0 with HCl)Sample rocked gently for 5 minutes then the residual ethanolamineremoved by dialysis or chromatography using pH 7.5 phosphate-NaClbuffer.

Example 15. Reaction of the medicant-azlactone product with an antibody.The azlactone derivative generated in Example 14 (note that other aminoacids can be used in place of glycine) is then reacted with the desiredpeptide or protein or other compound containing a primary amino group ata 1:1 ratio in buffer (25 mM sodium phosphate, 150 mM sodium chloride,pH 7.5) with gentle rocking at room temperature for 60 minutes. Thereaction is terminated by the addition of 1.0 mL of 25 mM ethanolamine(titrated to pH 9.00) with rocking for 5 minutes at room temperature).The drug-azlactone-ligand product can be purified by columnchromatography or dialysis to remove the ethanolamine by-product.

Azlactone linker to bind Illudin2 to a Glycopeptide. In an embodiment ofthe present invention, an illudin2 moiety 1302 linked via an azlactonelinker 1230 to a glycopeptide 1170 with biological activity binds toreceptors for the glycopeptide 1170 and directs the illudin2 moiety 1302to cell populations expressing the receptor. In an embodiment of thepresent invention, a glycopeptide 1170 linked via an azlactone linker1230 to an illudin2 moiety 1302 acts as an AM for the glycopeptidereceptor and directs the illudin2 moiety 1302 to tissues containingcells expressing the receptor. In an embodiment of the presentinvention, a glycopeptide 1170 linked via an azlactone linker 1230 to anilludin2 moiety 1302 acts as an AM for the glycopeptide receptor anddirects the illudin2 moiety 1302 to tumors containing cells expressingthe receptor. In an embodiment of the present invention, a glycopeptide1170 linked via an azlactone linker 1230 to an illudin2 moiety 1302 actsas an AM for the glycopeptide receptor and directs the illudin2 moiety1302 to tumors containing cells over-expressing the receptor compared tonon tumor cells

Azlactone linker to bind Illudin2 to a Lipid. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via an azlactonelinker 1230 to a lipid 1180 with biological activity binds to receptorsfor the lipid 1180 and directs the illudin2 moiety 1302 to cellpopulations expressing the lipid. In an embodiment of the presentinvention, a lipid 1180 linked via an azlactone linker 1230 to anilludin2 moiety 1302 acts as an AM for the lipid receptor and directsthe illudin2 moiety 1302 to tissues containing cells expressing thereceptor. In an embodiment of the present invention, a lipid 1180 linkedvia an azlactone linker 1230 to an illudin2 moiety 1302 acts as an AMfor the lipid receptor and directs the illudin2 moiety 1302 to tumorscontaining cells over-expressing the receptor compared to non tumorcells.

Azlactone linker to bind Illudin2 to a Peptide. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via an azlactonelinker 1230 to a peptide 1190 with biological activity binds to thepeptide receptor and directs the illudin2 moiety 1302 to cellpopulations expressing the receptor. In an embodiment of the presentinvention, a peptide 1190 linked via an azlactone linker 1230 to anilludin2 moiety 1302 acts as an AM for the peptide receptor and directsthe illudin2 moiety 1302 to tissues containing cells expressing thereceptor. In an embodiment of the present invention, a peptide 1190linked via an azlactone linker 1230 to an illudin2 moiety 1302 acts asan AM for the peptide receptor and directs the illudin2 moiety 1302 totumors containing cells expressing the receptor. In an embodiment of thepresent invention, a peptide 1190 linked via an azlactone linker 1230 toan illudin2 moiety 1302 acts as an AM for the peptide receptor anddirects the illudin2 moiety 1302 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

Azlactone linker to bind Illudin2 to a Steroid. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via an azlactonelinker 1230 to a steroid 1140 binds to receptors for the steroid anddirects the illudin2 moiety 1302 to cell populations expressing thereceptor. In an embodiment of the present invention, a steroid 1140linked via an azlactone linker 1230 to an illudin2 moiety 1302 acts asan AM for the steroid hormone receptor and directs the illudin2 moiety1302 to tissues containing cells expressing the receptor. In anembodiment of the present invention, a steroid 1140 linked via anazlactone linker 1230 to an illudin2 moiety 1302 acts as an AM for thesteroid hormone receptor and directs the illudin2 moiety 1302 to tumorscontaining cells expressing the receptor. In an embodiment of thepresent invention, a steroid 1140 linked via an azlactone linker 1230 toan illudin2 moiety 1302 acts as an AM for the steroid hormone receptorand directs the illudin2 moiety 1302 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

Azlactone linker to bind an Antibody to a Protein Toxin. In anembodiment of the present invention, an antibody 1110 is bound to aprotein toxin 1330 with an azlactone linker 1230. In an embodiment ofthe present invention, an antibody 1110 directs the protein toxin 1330to cell populations expressing the receptor. In an embodiment of thepresent invention, the antibody 1110 with an azlactone linker 1230 to aprotein toxin 1330 acts as an AM for a receptor and directs the proteintoxin 1330 to tissues containing cells expressing the receptor. In anembodiment of the present invention, an antibody 1110 with an azlactonelinker 1230 to the protein toxin 1330 acts as an AM for a receptor anddirects the protein toxin 1330 to tumors containing cells expressing thereceptor. In an embodiment of the present invention, an antibody 1110with an azlactone linker 1230 to the protein toxin 1330 acts as an AMfor a receptor and directs the protein toxin 1330 to tumors containingcells over-expressing the receptor compared to non tumor cells.

Azlactone linker to bind an Growth Factor to a Protein Toxin. In anembodiment of the present invention, a growth factor 1120 is bound to aprotein toxin 1330 with an azlactone linker 1230. In an embodiment ofthe present invention, the growth factor 1120 directs the protein toxin1330 to cell populations expressing the receptor to the growth factor1120. In an embodiment of the present invention, the growth factor 1120with an azlactone linker 1230 to a protein toxin 1330 acts as an AM fora receptor to the growth factor 1120 and directs the protein toxin 1330to tissues containing cells expressing the receptor. In an embodiment ofthe present invention, the growth factor 1120 with an azlactone linker1230 to the protein toxin 1330 acts as an AM for the growth factorreceptor and directs the protein toxin 1330 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

Azlactone linker to bind an Antibody to a Medicant. In an embodiment ofthe present invention, an antibody 1110 is bound to a medicant 1350 withan azlactone linker 1230. In an embodiment of the present invention, anantibody 1110 directs the medicant 1350 to cell populations expressingthe receptor. In an embodiment of the present invention, the antibody1110 with an azlactone linker 1230 to the medicant 1350 acts as an AMfor a receptor and directs the medicant 1350 to tissues containing cellsexpressing the receptor. In an embodiment of the present invention, anantibody 1110 with an azlactone linker 1230 to the medicant 1350 acts asan AM for a receptor and directs the medicant 1350 to tumors containingcells expressing the receptor. In an embodiment of the presentinvention, an antibody 1110 with an azlactone linker 1230 to themedicant 1350 acts as an AM for a receptor and directs the medicant 1350to tumors containing cells over-expressing the receptor compared to nontumor cells.

Azlactone linker to bind an Growth Factor to a Medicant. In anembodiment of the present invention, a growth factor 1120 is bound to amedicant 1350 with an azlactone linker 1230. In an embodiment of thepresent invention, the growth factor 1120 directs the medicant 1350 tocell populations expressing the receptor to the growth factor 1120. Inan embodiment of the present invention, the growth factor 1120 with anazlactone linker 1230 to a medicant 1350 acts as an AM for a receptor tothe growth factor 1120 and directs the medicant 1350 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, the growth factor 1120 with an azlactone linker 1230to the medicant 1350 acts as an AM for a receptor and directs themedicant 1350 to tumors containing cells expressing the receptor for thegrowth factor 1120. In an embodiment of the present invention, thegrowth factor 1120 with an azlactone linker 1230 to the medicant 1350acts as an AM for a receptor and directs the medicant 1350 to tumorscontaining cells over-expressing the receptor compared to non tumorcells.

Example 16. Synthesis of Medicant 114. (CH₃)₃S(O)I (110 mg, 0.4 mmol)and tBuOK(50 mg, 0.4 mmol) were dissolved in anhydrous DMSO (1 mL) andstirred at room temperature for 40 minutes at room temperature. Thenanalog 10 (50 mg, 0.2 mmol) in 1.0 mL of DMSO was added via syringe, andstirred for 3 hours. Reaction quenched with saturated NH₄Cl (1 mL),extracted with CH₂Cl₂, dried over Na₂SO₄, concentrated thenchromatographed (2:3 hexane:ethyl acetate) to yield analog 114 (20 mg.,50% yield).

Example 17. Synthesis of Medicant 115. Analog 10 (40 mg) and NAHCO₃ (50mg) are dissolved in 10 mL of 1:1 Ethanol and water mixture, thenhydroxylamine hydrochloride (20 mg) is added, stirred for 30 minutes atroom temperature. Water and ethyl acetate (1:1 mixture) is added,stirred, the organic layer is recovered, washed with saturated NaHCO₃and then brine, dried over Na₂SO₄, concentrated then chromatographed(2:3 ethyl acetate:hexane) to yield analog 115.

Example 18. Synthesis of Medicant 116. SeO₂ (45 mg) and 500 mg SiO2transferred into a dried RB flask, 5 mL of CH₂Cl₂ added, and stirred for1 hour under nitrogen. Then 250 μL of tBuO₂H added and stirred for 15minutes. Then 100 mg of Irofulven in 1 mL CH₂Cl₂ is added, and stirredfor 3 hours at room temperature under a nitrogen atmosphere. Product isfiltered, wash twice with water (25 mL), twice with brine (25 mL), driedover Na₂SO₄ and concentrated then chromatographed (4:1 hexane:ethylacetate) to yield analog 116.

Example 19. Synthesis of Medicant 116. Analog 117: Illuclin S (100 mg,0.378 mmol) and glutaric anhydride (215.46 mg, 1.89 mmol) are dissolvedin 5 mL of CH₂Cl₂, and DMAP added (92.23 mg, 0.756 mmol), and stirredfor 2 hours at room temperature. The CH₂Cl₂ is evaporated, 5 mL of wateris added, and stirred for 1 hour. The solution is extracted with 10 mLof CH₂Cl₂, washed with water, dried over Na₂SO₄ and concentrated toyield analog 117 (120 mg).

Example 20. Synthesis of Analog 118: Analog 302 (75 mg), glutaricanhydride (20 mg) are dissolved in 5 mL of CH₂Cl₂, and DMAP added (42mg), and stirred for 2 hours at room temperature. The CH₂Cl₂ isevaporated, 5 mL of water added, and stirred for 1 hour. Solution isextracted with 10 mL of CH₂Cl₂, washed with water, dried over Na₂SO₄ andconcentrated to yield analog 118 (120 mg).

Example 21. Synthesis of Analog 119: Analog 114 (10 mg) is dissolved in1.5 mL of acetone with 1.0 mL of 4N H₂SO₄, and contents stirred for 1.5hours at room temperature. Then 10 mL of CH₂Cl₂ and 10 mL of water areadded, extracted, and the organic layer recovered which is then washedwith saturated NaHCO₃ and saline, dried over Na₂SO₄ and concentrated,and analog 119 recovered (analog 128 is a byproduct).

Example 22. Synthesis of Analog 120: Analog 10 (50 mg), NaHCO₃ aredissolved in 10 mL of 1:1 mixture of water and ethanol, then NH₂NH₂ (0.5mL added with stirring at room temperature for one hour. The solution isextracted with CH₂Cl₂ twice, the organic layer recovered, washed withwater, then NaHCO₃ solution, dried over Na₂SO₄, and evaporated to yieldanalog 120 (30 mg).

Example 23. Synthesis of Analog 121: Analog 10 (50 mg) and NaCO₂CH₃ (75mg) are dissolved in 10 mL of 1:1 mixture of water and ethanol 1:1, thensemicarbazide hydrochloride salt (H₂NNHCONH₂HCl, 50 mg) added, andstirred for 2 hours at room temperature. The solution is extracted withCH₂Cl₂ twice, the organic layer recovered, washed with water, thenNaHCO₃ solution, dried over Na₂SO₄, and evaporated then chromatographed(5% methanol in ethyl acetate) to yield analog 121.

Example 24. Synthesis of Analog 122: Analog 10 (50 mg) and NaCO₂CH₃ (75mg) are dissolved in 5 mL of ethanol, then phenylhydrazide (50 mg) isadded, stirred for 1 hour at room temperature. Then 5 mL of water isadded, followed by extraction with ethyl acetate, washed with water,dried over Na₂SO₄ and concentrated and chromatographed (5% methanol inethyl acetate) to yield analog 122.

Example 25. Synthesis of Analog 123: Analog 10 (50 mg) and NaCO₂CH₃ (75mg) are dissolved in 10 mL of 1:1 water and ethanol, then H₂NNHTS(H₂NNHS(═O)₂(phenyl)methyl, 50 mg) is added, stirred for 2 hour at roomtemperature. Then 5 mL of water is added, followed by extraction withethyl acetate, washed with water, dried over Na₂SO₄ and concentrated andchromatographed (5% methanol in ethyl acetate) to yield analog 123.

Example 26. Synthesis of Analog 124: Analog 115 (15 mg) and NaOAc(15 mg)are dissolved in acetic anhydride (1 mL) and stirred for 2 hours, thensodium acetate (300 mg) is added with stirring for 1 hour. Then themixture is chromatographed (10% ethyl acetate in hexane) to give analog124.

Example 27. Synthesis of Analog 125: Analog 10 (50 mg) and NaCO₂CH₃ (75mg) are dissolved in 5 mL of ethanol, then the dinitrophenylhydrazide(50 mg) is added, stirred for 1 hour at room temperature. Then 5 mL ofwater is added, followed by extraction with ethyl acetate, washed withwater, dried over Na₂SO₄ and concentrated and chromatographed (5%methanol in ethyl acetate) to yield analog 125.

Example 28. Synthesis of Analog 126: Analog 11 (40 mg), hydroxylamine(20 mg), NaHCO₃ (50 mg) are dissolved in 10 mL of ethanol and water(1:1) then stirred at room temperature for 90 minutes. Then the mixtureis extracted with water (10 mL) and ethyl acetate (20 mL), the organiclayer washed with saturated NaHCO₃ then brine, dried over Na₂SO₄ andconcentrated, then chromatographed (2:3 ethyl acetate:hexane) to giveanalog 126.

Example 29. Synthesis of Analog 127: Analog 10 (100 mg) and NH₄Cl (1.5equivalent) are dissolved in 1,4-dioxane (5 mL) and water (0.2 mL), thenNaCN added (1.3 equivalents), stirred for 1 hour at room temperature.Then ethyl ether (20 mL) was added, the organic layer recovered, washedwith water, washed with brine, then dried over Na₂SO₄, thenchromatographed (2:3 ethyl acetate:hexane) to yield analog 127.

Example 30. Synthesis of Analog 128: Analog 114 (10 mg) is dissolved in1.5 mL of acetone with 1.0 mL of 4N H₂SO₄, and contents stirred for 1.5hours at room temperature. Then 10 mL of CH₂Cl₂ and 10 mL of water areadded, extracted, and the organic layer recovered which is then washedwith saturated NaHCO₃ and saline, dried over Na₂SO₄ and concentrated,and analog 128 recovered (analog 119 is a byproduct).

Example 31. Synthesis of Analog 129: Acylfulvene (200 mg) is dissolvedin anhydrous THF (10 mL) at room temperature then NaBH₄ (100 mg) isadded slowly for 30 minutes. Reaction is quenched with 1 mL of waterthen extracted with ethyl acetate (10 mL), washed with saturated NaHCO₃,and dried over Na₂SO₄, then concentrated to yield analog 129. If need bethe compound can be purified by chromatography (1:1 ethylacetate:hexane).

Example 32. Analog 141: Analog 129 (200 mg) is dissolved in CH₂Cl₂ atroom temperature, then 1,4-dimethyl but-2-ynedioate (1.1 equivalent) isadded slowly and mixture allowed to react for only hour, then evaporatedto yield analog 141. If need be the compound can be purified bychromatography (1:1 ethyl acetate:hexane).

Example 33. Synthesis of Analog 142: Analog 141 (100 mg) is dissolved inCH₂Cl₂ at room temperature then Dess-Martin Periodinane reagent (200 mg)added with stirring for 1 hour to yield analog 142. If need be thecompound can be purified by chromatography (1:1 ethyl acetate:hexane).

Example 34. Synthesis of Analog 146: Analog 127 (35 mg, 0.117 mmol),DMAP (5 mg), and diimidazole (22 mg, 1.2 eq) were dissolved in anhydrousCH₂Cl₂ under an argon atmosphere, and stirred for 30 minutes. Thesolution was cooled to 20° C. then tributyl tin hydride (Bu₃SnH, 0.6 mL)and azobis isobutylnitrite (4 mg) were added with stirring for 30minutes. The mixture was filtered then chromatographed (1:10 ethylacetate:hexane) to remove impurities and starting materials, thenchromatographed (2:3 ethyl acetate:hexane) to yield analog 146.

Example 35. Synthesis of Analog 147: Irofulven (10 mg) is dissolved in 3mL of acetone and 1 M H₂SO₄ solution (1:1) with stirring at roomtemperature and 2-Mercaptobenzothiazole (1 equivalent) is added, stirredfor 2 hours, then partitioned between ethyl acetate and water. Theorganic extract is washed with saturated NaHCO₃ and saline untilneutral, dried over MgSO₄, concentrated then chromatographed (1:1 ethylacetate:hexane) to give analog 147.

Example 36. Synthesis of Analog 148: Irofulven (10 mg) is dissolved in 3mL of acetone and 1 M H₂SO₄ solution (1:1) with stirring at roomtemperature and 2-Mercaptobenzoxazole (1 equivalent) is added, stirredfor 2 hours, then partitioned between ethyl acetate and water. Theorganic extract is washed with saturated NaHCO₃ and saline untilneutral, dried over MgSO₄, concentrated then chromatographed (1:1 Ethylacetate:hexane) to give analog 148.

Example 37. Synthesis of Analog 149: Irofulven (10 mg) is dissolved in 4mL of acetone and 1 M H₂SO₄ solution (1:1) with stirring at roomtemperature and thiol-imidazole (1 equivalent) is added, stirred for 24hours, then partitioned between ethyl acetate and water. The organicextract is washed with saturated NaHCO₃ and saline until neutral, driedover MgSO₄, concentrated then chromatographed (1:1 ethyl acetate:hexane)to give analog 149.

Example 38. Synthesis of Analog 150: Irofulven (10 mg) is dissolved in 4mL of acetone and 1 M H₂SO₄ solution (1:1) with stirring at roomtemperature and 2-mercapto-5-methylbenzimidazole (1 equivalent) isadded, stirred for 12 hours, then partitioned between ethyl acetate andwater. The organic extract is washed with saturated NaHCO₃ and salineuntil neutral, dried over MgSO₄, concentrated then chromatographed (1:1ethyl acetate:hexane) to give analog 150.

Example 39. Synthesis of Analog 151: Irofulven (10 mg) is dissolved in 3mL of acetone and 1 M H₂SO₄ solution (1:1) with stirring at roomtemperature and 1-phenyl-1,2,3,4-tetraazole-5-thiol (1 equivalent) isadded, stirred for 2 hours, then partitioned between ethyl acetate andwater. The organic extract is washed with saturated NaHCO₃ and salineuntil neutral, dried over MgSO₄, concentrated then chromatographed (1:1ethyl acetate:hexane) to give analog 151.

Example 40. Synthesis of Analog 152: Irofulven (10 mg) is dissolved in 3mL of acetone and 1 M H₂SO₄ solution (1:1) with stirring at roomtemperature and 2-mercapto-5-nitro benzimidazole (1 equivalent) isadded, stirred for 2 hours, then partitioned between ethyl acetate andwater. The organic extract is washed with saturated NaHCO₃ and salineuntil neutral, dried over MgSO₄, concentrated then chromatographed (1:1ethyl acetate:hexane) to give analog 152.

Example 41. Synthesis of Analog 153: Irofulven (10 mg) is dissolved in 3mL of acetone and 1 M H₂SO₄ solution (1:1) with stirring at roomtemperature and 1,2,4-Triazole-3-thiol (1 equivalent) is added, stirredfor 2 hours, then partitioned between ethyl acetate and water. Theorganic extract is washed with saturated NaHCO₃ and saline untilneutral, dried over MgSO₄, concentrated then chromatographed (1:1 ethylacetate:hexane) to give analog 153.

Example 42. Synthesis of Analog 154: Irofulven (10 mg) is dissolved in 3mL of acetone and 1 M H₂SO₄ solution (1:1) with stirring at roomtemperature and 2-sulfanylpteridin-4-ol (1 equivalent) is added, stirredfor 2 hours, then partitioned between ethyl acetate and water. Theorganic extract is washed with saturated NaHCO₃ and saline untilneutral, dried over MgSO₄, concentrated then chromatographed (1:1 ethylacetate:hexane) to give analog 154.

Example 43. Synthesis of Analog 155: Irofulven (10 mg) is dissolved in 3mL of acetone and 1 M H₂SO₄ solution (1:1) with stirring at roomtemperature and 4-(5-sulfanyl-1H-1,2,3,4-tetrazol-1-yl)phenol (1equivalent) is added, stirred for 2 hours, then partitioned betweenethyl acetate and water. The organic extract is washed with saturatedNaHCO₃ and saline until neutral, dried over MgSO₄, concentrated thenchromatographed (1:1 ethyl acetate:hexane) to give analog 155.

Example 44. Synthesis of Analog 156: Irofulven (10 mg) is dissolved in 3mL of acetone and 1 M H₂SO₄ solution (1:1) with stirring at roomtemperature and 4-(5-sulfanyl-1-1,2,3,4-tetrazol-1-yl)benzoic acid (1equivalent) is added, stirred for 2 hours, then partitioned betweenethyl acetate and water. The organic extract is washed with saturatedNaHCO₃ and saline until neutral, dried over MgSO₄, concentrated thenchromatographed (1:1 ethyl acetate:hexane) to give analog 156.

Example 45. Synthesis of Analog 159: Illudin S (300 mg) is dissolvedacetic anhydride (6 mL) and stirred for 15 minutes, then sodium acetate(300 mg) is added with stirring for 1 hour. Water (6 mL) is added, ethylacetate extraction performed, washed with sodium bicarbonate solution,dried over Na₂SO₄, concentrated then chromatographed (2:3 ethylacetate:hexane) to give analog 159.

Example 46. Synthesis of Analog 160: Analog 159 (60 mg) is dissolved indry CH₂Cl₂ (6 mL) under nitrogen at room temperature and glutaricanhydride (100 mg) with DMAP (20 mg) is added with stirring for 30minutes. The solvent is removed, water added, extracted with CH₂Cl₂,washed with water, dried over Na₂SO₄, concentrated then chromatographed(2:3 ethyl acetate:hexane) to give analog 160.

Example 47. Synthesis of Analog 161: Dehydroilludin S (300 mg) isdissolved acetic anhydride (6 mL) and stirred for 15 minutes, thensodium acetate (300 mg) is added with stirring for 1 hour. Water (6 mL)is added, ethyl acetate extraction performed, washed with sodiumbicarbonate solution, dried over Na₂SO₄, concentrated thenchromatographed (2:3 ethyl acetate:hexane) to give analog 161.

Example 48. Synthesis of Analog 162: Dehydroilludin S (60 mg) isdissolved in dry CH₂Cl₂ (6 mL) under nitrogen at room temperature andglutaric anhydride (150 mg) with DMAP (50 mg) is added with stirring for30 minutes. The solvent is removed, water added, extracted with CH₂Cl₂,washed with water, dried over Na₂SO₄, concentrated then chromatographed(2:3 ethyl acetate:hexane) to give analog 162.

Example 49. Synthesis of Analog 163: Analog 159 (20.25 mg), DMAP (20 mg)are dissolved in dry CH₂Cl₂ (6 mL) at 0° C. under nitrogen atmosphereand stirred for 10 minutes. Then chloroacetyl chloride (0.2 mL) is addedslowly and the mixture stirred for 30 minutes, warmed to roomtemperature with stirring over 15 minutes. Then water (6 mL) is added,mixed, and then extracted with CH₂Cl₂. The organic layer is washed withsaturated NaHCO₃ followed by a saline wash, dried over Na₂SO₄ thenchromatographed (2:3 ethyl acetate:hexane) to yield analog 163 (60%yield).

Example 50. Synthesis of Analog 164: Irofulven (50 mg), DMAP (40 mg) aredissolved in dry CH₂Cl₂ (6 mL) at 0° C. under nitrogen atmosphere andstirred for 10 minutes. Then chloroacetyl chloride (0.2 mL) is addedslowly and the mixture stirred for 30 minutes, warmed to roomtemperature with stirring over 15 minutes. Then water (6 mL) is added,mixed, and then extracted with CH₂Cl₂. The organic layer is washed withsaturated NaHCO₃ followed by a saline wash, dried over Na₂SO₄ thenchromatographed (2:3 ethyl acetate:hexane) to yield analog 164 (60%yield).

Example 51. Synthesis of Analog 165: Analog 164 (40 mg) is dissolved indry CH₂Cl₂ (6 mL) at room temperature under nitrogen atmosphere andstirred for 10 minutes. Then 1 mL of morpholine is added drop wise, withstirring for 30 minutes. The reaction is diluted with water (6 mL),extracted with CH₂Cl₂ (12 mL). The organic layer is washed withsaturated NaHCO₃ then washed with saline, dried over Na₂SO₄ andchromatographed (2:3 ethyl acetate:hexane) to yield 165 (35% yield).

Example 52. Synthesis of Analog 166 and analog 167 (prepared together):Analog 160 (30 mg) is dissolved in methanol (4 mL) at 0° C., and 1NH₂SO₄ (1 mL) is added with stirring for 1 hour. Water (6 mL) is added,extracted with ethyl acetate, washed with NaHCO₃ then a brine solution,dried over MgSO₄, concentrated and then chromatographed (1:1 ethylacetate:hexane) to yield analogs 166 and 167 in equal amounts.

Example 53. Synthesis of Analog 168: Analog 162 (20 mg) is dissolved inmethanol (5 mL) at 0° C. and stirred for 10 minutes, then 1 mL of 1NH₂SO₄ in methanol is slowly added, followed by stirring for 30 minutes.Water is added, followed by an ethyl acetate extraction, washed withNaHCO₃ then a brine solution, dried over Na₂SO₄, concentrated thenchromatographed (1:1 ethyl acetate:hexane) to yield analog 168.

Example 54. Synthesis of Analog 169: Dehydroilludin S (20 mg), DMAP (20mg) are dissolved in dry CH₂Cl₂ (6 mL) at 0° C. under nitrogenatmosphere and stirred for 10 minutes. Then chloroacetyl chloride (0.2mL) is added slowly and the mixture stirred for 30 minutes, warmed toroom temperature with stirring over 15 minutes. Then water (6 mL) isadded, mixed, then extracted with CH₂Cl₂. The organic layer is washedwith saturated NaHCO₃ followed by a saline wash, dried over Na₂SO₄ thenchromatographed (2:3 ethyl acetate:hexane) to yield analog 169 (60%yield).

Example 55. Synthesis of Analog 176: To a solution of analog 9 (266umol), Boc protected leucine amino acid (300 umol) and DMAP(dimethylaminopyridine, 110 umol) in CH₂Cl₂ (2.5 mL) at 0° C. is addedDCC (dicyclohexylcarbodiimide; 1.0M in CH₂Cl₂, 300 umol)/. The mixtureis stirred for 35 minutes then 5 μl_, of water added to quench thereaction. The mixture is diluted with hexane and precipitate filteredoff, solvent evaporated off and crude product chromatographed (2:1hexanes-ethyl acetate) to give the desired Boc-protected derivative of176 at 80% yield. The Boc group is removed by dissolving theBoc-protected derivative in a 1:1 mixture (2.0 mL) of 1,4-dioxane and 2MH₂SO₄, stirred for 18 hours, then partitioned between ethyl acetate andwater. Aqueous layer is extracted with ethyl acetate and extractsdiscarded. Aqueous layer is neutralized with saturated NaHCO₃ andextracted again with ethyl acetate. Organic layer is washed with brine,dried with MgSO₃, solvent evaporated to yield the analog 9 amino acidderivative. As the amine derivative is unstable over prolonged periodsof time it can be converted to the very stable trifluoroacetate salt bydissolving in CH₂Cl₂ adding the equal molar amount of trifluoroaceticacid and concentrating to dryness.

Example 56. Synthesis of Analog 178: Analog 9 (15 mg) is dissolved inCH₂Cl₂ (2.0 mL) under a nitrogen atmosphere at room temperature,succinic anhydride (1 equivalent) is added, followed by DMAP (10 mg) andstirring for 30 minutes. Solvent is removed and product recrystallizedto give analog 178.

Example 57. Synthesis of Analog 179: To a solution of Analog 9 (266μmol), Boc protected glycine amino acid (300 umol) and DMAP(dimethylaminopyridine, 110 umol) in CH₂Cl₂ (2.5 mL) at 0° C. is addedDCC (dicyclohexylcarbodiimide; 1.0M in CH₂Cl₂, 300 umol)/. The mixtureis stirred for 35 minutes then 5 μl_, of water added to quench thereaction. The mixture is diluted with hexane and precipitate filteredoff, solvent evaporated off and crude product chromatographed (2:1hexanes-ethyl acetate) to give the desired Boc-protected derivative of179 at 80% yield. The Boc group is removed by dissolving theBoc-protected derivative in a 1:1 mixture (2.0 mL) of 1,4-dioxane and 2MH₂SO₄, stirred for 18 hours, then partitioned between ethyl acetate andwater. Aqueous layer is extracted with ethyl acetate and extractsdiscarded. Aqueous layer is neutralized with saturated NaHCO₃ andextracted again with ethyl acetate. Organic layer is washed with brine,dried with MgSO₃, solvent evaporated to yield the analog 9 amino acidderivative. As the amine derivative is unstable over prolonged periodsof time it can be converted to the very stable trifluoroacetate salt bydissolving in CH₂Cl₂ adding the equal molar amount of trifluoroaceticacid and concentrating to dryness.

Example 58. Synthesis of Analog 180: Illudin M (50 mg) is dissolved indry benzene (10 mL) under a nitrogen atmosphere, and vanadylacetylacetonate (VO(acac)₂, 1.2 mg) is added. Then t-butyl hydroperoxide(t-BuO₂H, 0.5 mL) in benzene is added drop wise with stirring for 30minutes. A saturated solution of Na₂S₂O₃ is added (10 mL), thenextraction with ethyl acetate, and the organic layer is dried overNa2SO₄, concentrated then chromatographed) (1:1 ethyl acetate:hexane) togive analog 180.

Example 59. Synthesis of Analog 181: Analog 159 (40 mg) was dissolved indry benzene (8 mL) under a nitrogen atmosphere, and vanadylacetylacetonate (VO(acac)₂, 2 mg) was added. Then t-butyl hydroperoxide(t-BuO₂H, 0.5 mL) in benzene was added drop wise with stirring for 30minutes. A saturated solution of Na₂S₂O₃ is added (10 mL), thenextraction with ethyl acetate, followed by a brine wash, and the organiclayer was then dried over Na₂SO₄, concentrated then chromatographed)(1:1 ethyl acetate:hexane) to give analog 181.

Example 60. Synthesis of Analog 189: To a solution of Irofulven (1.00equivalent), maleimide (1.71 equivalent), triphenylphosphine (PPh₃, 1.71equivalent) in 1.5 mL of THF at −40° C., is added DEAD(diethylazodicarboxylate; 1.68 equivalent). The mixture is stirred for30 minutes then water (20 μL) added to quench the reaction. The mixtureis concentrated on a rotary evaporator and crude product ischromatographed on a silica column (10:3 hexanes:ethyl acetate) to yieldan orange compound (20% yield).

Example 61. Synthesis of Analog 190: To a solution of analog 9(6-hydroxy-n-propylacylfulvene structure below, 1.00 equivalent),maleimide (1.23 equivalent), triphenylphosphine (PPh₃, 1.13 equivalent)in 2.5 mL, of THF at −40° C., is added DIAD (diisopropylcarbodiimide;1.44 equivalent). The mixture is stirred for 1 hour then water (10 μL)added to quench the reaction. The mixture is concentrated on a rotaryevaporator and crude product is chromatographed on a silica column (5:1→10:3 hexanes:ethyl acetate) to yield an orange compound (15% yield).

Example 62. Synthesis of Analog 196: To a solution of analog 9 (266umol), Boc protected proline amino acid (300 umol) and DMAP(dimethylaminopyridine, 110 umol) in CH₂Cl₂ (2.5 mL) at 0° C. is addedDCC (dicyclohexylcarbodiimide; 1.0M in CH₂Cl₂, 300 umol)/. The mixtureis stirred for 35 minutes then 5 μL of water added to quench thereaction. The mixture is diluted with hexane and precipitate filteredoff, solvent evaporated off and crude product chromatographed (2:1hexanes-ethyl acetate) to give the desired Boc-protected derivative of196 at 80% yield. The Boc group is removed by dissolving theBoc-protected derivative in a 1:1 mixture (2.0 mL) of 1,4-dioxane and 2MH₂SO₄, stirred for 18 hours, then partitioned between ethyl acetate andwater. Aqueous layer is extracted with ethyl acetate and extractsdiscarded. Aqueous layer is neutralized with saturated NaHCO₃ andextracted again with ethyl acetate. Organic layer is washed with brine,dried with MgSO₃, solvent evaporated to yield the analog 9 amino acidderivative. As the amine derivative is unstable over prolonged periodsof time it can be converted to the very stable trifluoroacetate salt bydissolving in CH₂Cl₂ adding the equal molar amount of trifluoroaceticacid and concentrating to dryness.

Example 63. Synthesis of Analog 198: Irofulven (26.3 mg, 107 umol),p-nitrophenol (16.2 mg, 116 umol) and PPh3 (30.8 mg, 117 umol) weredissolved in anhydrous THF (1.5 mL) at −40° C., the DEAD (25 μL, 160umol) was added, followed by stirring for 30 minutes, then diluted withhexane. The precipitate was filtered off, solvent evaporated, and crudeproduct chromatographed (6:1->2:1 hexane:ethyl acetate) to give analog198 as a yellow product (18.5 mg, 47%).

Example 64. Analogs 199 and 200 (prepared together): Irofulven (25.2 mg,102 umol), phenol (11.5 mg, 122 umol) and PPh₃ (29.1 mg, 117 μmol) weredissolved in anhydrous THF (1.0 mL) at −40° C., the DEAD (25 μL, 192μmol) was added, followed by stirring for 30 minutes, then diluted withhexane. The precipitate was filtered off, solvent evaporated, and crudeproduct chromatographed (6:1->3:1 hexane:ethyl acetate) to give analog199 (8.2 mg, 25%) and analog 200 (14.6 mg, 44%) as a yellow products.

Example 65. Synthesis of Analog 201 [6-(acetamidopropyl)acylfulvenel: Toa solution of analog 195 (49.1 umol) and water (20 μL in THF (0.5 ml)was added a solution of O-acetyl-2-(diphenylphosphino)phenol (39.0 umol)in THF (0.5 mL). The mixture was stirred for 3 days at room temperaturethen concentrated. The crude product was chromatographed (100% ethylacetate) to yield 8.2 mg of analog 201.

Example 66. Synthesis of Analog 202 (i.e., analog 211 linked toproline): Prepared via Staudinger ligation. To a solution of analog 195(94 umol) in THF (1.2 mL), water (40 μL) was added, the was addedN-Boc-proline, 2-(diphenylphosphino)phenyl ester (101 μmol) in THF (0.8mL). The mixture was stirred for 3 days at room temperature thenconcentrated. The crude product was chromatographed (5:1->1:2hexanes-ethyl acetate) to yield 31.4 mg (66.7 umol) of analog 202-Boc(71%). The analog 202-Boc was dissolved (66.7 umol) in dioxane (2.0 mL)and 2.0 mL of 2M H₂SO₄ was added, and the mixture was stirred overnight.Water and ethyl acetate was added, orange color appeared in the aqueous.The aqueous was extracted again with ethyl acetate and organic layerdiscarded. Sodium bicarbonate was added to aqueous until basic,re-extracted with ethyl acetate. The solution was dried with magnesiumsulphate, concentrated to dryness, dissolved in CH₂Cl₂ and 8 mg of TFAadded (1 drop). Analog 202 was obtained in an amount of 22.2 mg (69%).

Example 67. Synthesis of Analog 203: Synthesis of Analog 208 (9.2 mg,16.5 umol) is dissolved in CH₂Cl₂ (1.5 mL), 1 drop of anisole added,then 0.5 mL of trifluro acetic acid for 15 minutes. The mixture isconcentrated, dissolved in water, then re-extracted with CH₂Cl₂, and theorange color remains in the aqueous phase, which is concentrated to giveanalog 203 as the orange colored TFA salt (10.0 mg).

Example 68. Synthesis of Analog 204: Although the Fmoc-Pro-OH wouldpreferentially react with the primary hydroxyl group on Illudin S, theresulting ester linkage is not stable, as illudin S was recovered afterstorage in CDCl₃ for several days at room temperature. The secondaryhydroxy group of illudin S was therefore used for coupling withpeptides. The primary hydroxy group of illudin S first protected with aTBS group (TBSCl, Imidazole, and DMF, 92%) to produce analog 204.

Example 69. Synthesis of Analog 205 Analog 309 (20 mg, 0.050 mmol, 1equiv.), triphenylphosphine (40 mg, 0.1525 mmol, 3 equiv.) was dissolvedin THF (1 mL) at room temperature. After 20 hours a few drops of waterwas added and the mixture was heated up at 70° C. After 5 hours thesolution was cooled down and evaporated. The residue was chromatographed(hexane/EtOAc/Et₃N 4:1:0.1) to give analog 205 (5.3 mg, 29%) as an oil.

Example 70. Synthesis of Analog 206: Analog 205 (14 mg, 0.037 mmol, 1equiv.) was dissolved in CH₃CN (0.5 mL) and pyridine (0.1 mL) at 0° C.To this solution was added HF.Pyridine (7 μL, 0.245 mmol, 35 M, 6.6equiv.). After 10 min K₂CO₃ (0.5 mL, 0.5 M) was added and this mixturewas chromatographed (CH₂Cl₂/Methanol/Et₃N 5:0.5:0.1) to give analog 206(10 mg, 68%) as an oil.

Example 71. Synthesis of Analog 207 (211-leucine): Prepared viaStaudinger ligation. To a solution of analog 195 (101 umol) in THF (1.0mL), water (40 μL) was added, then was added N-Boc-leucine,2-(diphenylphosphino)phenyl ester (95.9 μmol) in THF (1.2 mL). Themixture was stirred for 6 days at room temperature then concentrated.The crude product was chromatographed (1:1 hexanes-ethyl acetate) toyield 27.3 mg of analog 207-Boc. The analog 207-Boc was dissolved (16μmol) in CH₂Cl₂ with 3 drops of anisole, TFA was added (0.3 mL), and themixture was stirred for 15 minutes then concentrated. The crude materialwas dissolved in water then extracted with CH₂Cl₂. The aqueous layer wasrecovered and concentrated to yield 17.4 mg of the analog 207 TFA salt(87%).

Example 72. Analog 208: The TFA salt of analog 196 (13.7 mg, 28.2 μmol)was dissolved in anhydrous DMF (2.5 mL), Boc-Serine-OH (9.6 mg, 47 umol)was added, ODHBT (13.0 mg, 79.4 umol), cooled to 0° C. under a nitrogenatmosphere. Next EDC (15.1 mg) was added followed by NMM (10 μL) toadjust pH, and the mixture stirred at 0° C. for 3 hours. The reactionwas added to ethyl acetate/water mixture, and the orange productappeared in the organic layer. The aqueous layer was re-extracted withethyl acetate, organic layers combined, washed with dilute NaHSO₄,water, saturated NaHCO₃, brine, then dried with MgSO₄. The organic layerwas concentrated then chromatographed (1:3 hexane:ethyl acetate) toyield analog 208 as an orange residue (63% yield).

Example 73. Synthesis of Analog 209: The TFA salt of analog 196 (12.5mg, 25.7 μmol) was dissolved in anhydrous DMF (2.5 mL), Boc-Serine-SerOH (88.6 umol) was added, ODHBT (33.9 mg, 205 umol), cooled to 0° C.under a nitrogen atmosphere. Next EDC (142 umol) was added followed byNMM (10 μL) to adjust pH, and the mixture stirred at 0° C. but allowedto gradually warm as the ice melts. The mixture was stirred a total of16 hour then 1 mL water added followed by stirring for 50 minutes. Thereaction was added to ethyl acetate/water mixture, and the orangeproduct appeared in the organic layer. The aqueous layer wasre-extracted with ethyl acetate, organic layers combined, washed withdilute NaHSO₄, water, saturated NaHCO₃, brine, and then dried withMgSO₄. The organic layer was concentrated then chromatographed (10:1ethyl acetate: methanol) to give analog 209 as an orange residue (5.9mg, 36% yield).

Example 74. Synthesis of Analog 210(Ac-Hyp-Ser-Ser-Chg-Gln-Ser-Ser-Pro-O—(CH₂)₃-acylfulvene): To a mixtureof Analog 196 TFA salt (21.6 umol), the peptideAc-Hyp-Ser-Ser-Chg-Gln-Ser-Ser-OH (30.3 umol), ODHBt(3,4,-dihydroxy-4-oxo-1,2,3-benzo-triazine-3-yl ester, 71.7 μmol) andNMM (N-methylmorpholine; 7.5 ul) in DMF (2.0 ml) at room temperature isadded EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,68 μmol), the mixture stirred for 2 hours at room temperature, thendiluted with 10 mL of water. Solution is directly chromatographed on areverse phase C18 column (4:1->2:1, water/acetonitrile gradient) toyield 69% of analog 210.

Example 75. Synthesis of Analog 212 (Illudin M-proline) Illudin M (20mg, 0.081 mmol, 1 equivalent), DMAP (1 mg, 0.008 mmol, 0.1 equiv.) andFmoc-Pro-OH (33 mg, 0.097 mmol, 1.2 equiv.) were dissolved in CH₂Cl₂ (1mL) at 0° C., to which was added a CH₂Cl₂ solution of DCC (100 μL, 0.1mmol, 1 M, 1.2 equiv.). The temperature of the mixture gradually rose to5° C. in 1.5 hours and then the mixture was filtered through a pad ofCelite. The filtrate was concentrated and the residue waschromatographed (CH₂Cl₂/EtOAc 5:0.1-5:0.4) to giveIlludin-M-proline-Fmoc protected analog (36 mg, 79%) as oil. The protonspectra of this oil showed that it was a mixture of two isomers(rotamers). And then this oil was dissolved in CH₂Cl₂ (4 mL) and treatedwith piperidine (1 mL) at 0° C. After 0.5 hours the solution wasconcentrated and the concentrate was chromatographed (CH₂Cl₂/Methanol5:0.4) to give analog 212 (15 mg, 54%) as oil.

Example 76. Synthesis of Analog 213: Analog 204 is coupled withFmoc-Pro-H (DMAP, CH₂Cl₂, DCC, 0° C., 85%), followed by deprotection ofFmoc group with 20% piperidine in CH₂Cl₂ to produce analog 213 in 78%yield.

Example 77. Synthesis of Analog 214 (Illudin S-Pro-Ser-Ser-HHOAc): TheFmoc protected peptide of H-Ser-Ser-OH was prepared by takingH-Ser-Ser-OH (50 mg, 0.26 mmol, 1 equiv.) and K₂CO₃ (89.7 mg, 0.65 mmol,2.5 equiv.), dissolving in a mixture of water (4 mL) and dioxane (3 mL)at 0° C. To this solution FmoCl (67.3 mg, 0.26 mmol, 1 equiv.) was addedin several portions. After 18 hours the mixture was acidified by KHSO₄and the pH raised to 2.5. Then this mixture was taken up by ethylacetate, which was washed with brine, dried, filtered and evaporated.The residue was chromatographed (CH₂Cl₂/Methanol/HOAc 5:1:0.1) to give3.27 (75 mg, 70%) as a white solid. The analog 212 (Illudin Stosylate-Pro) (42.8 mg 0.09 mmol, 0.9 equiv.), and the Fmoc protectedH-Ser-Ser-OH peptide (41.2 mg, 0.1 mmol, 1 equiv.) were dissolved in DMF(1.5 mL) at 0° C. To this solution was added NMM (22 μL, 0.2 mmol, 2equiv.), ODHBt (29.4 mg, 0.18 mmol, 1.8 equiv.), and EDC (31.1 mg, 0.16mmol, 1.6 equiv.). The solution temperature was then raised to roomtemperature and kept for 3 hours before it was taken up by ethylacetate. The mixture was then washed with saturated sodium bicarbonateand brine. It was then dried, filtered and evaporated. The residue waschromatographed (CH₂Cl₂/Methanol 5:0.3) to give analog 214 (50.5 mg,67%) as an oil.

Example 78. Synthesis of Analog 215: (IlludinS-Pro-Ser-Ser-Gln-Chg-Ser-Ser-Hyp-Ac) Analog 204 is coupled withFmoc-Pro-H (DMAP, CH₂Cl₂, DCC, 0° C., 85%), followed by deprotection ofFmoc group with 20% piperidine in CH₂Cl₂ to produce analog 213 in 78%yield. Peptide conjugate, analog 215 was obtained from further couplingwith hepta-peptide Ac-Hyp-Ser-Ser-Chg-Gln-Ser-Ser-OH (ODHBt, NMM, DMF,0° C., 47%).

Example 79. Synthesis of Analog 216: (IlludinM-Pro-Ser-Ser-Gln-Chg-Ser-Ser-Hyp-Ac). Analog 212 was further coupledwith the commercially available hepta-peptideAc-Hyp-Ser-Ser-Chg-Gln-Ser-Ser-OH (ODHBt, NMM, DMF, EDC, 0° C.) to yieldanalog 216 at 33%. The low yield resulted from repeated chromatographicpurification as the purity of the final raw product was estimated byHPLC to be only 70%.

Example 80. Synthesis of Analog 217: To a solution of Irofulven (1.00equivalent), epsilon-maleimidocaproic acid (1.27 equivalent), DMAP (0.15equivalent) in 1.0 mL of methylene chloride (CH₂Cl₂) at 0° C., is addedDCC (dicyclohexylcarbodiimide; 1.27 equivalent) in methylene chloride(CH₂Cl₂). The mixture is stirred for 1.25 hours, diluted with hexane andprecipitated is filtered. Residual solvent is evaporated off, and oilresidue is chromatographed on a silica column (2:1 hexanes:ethylacetate) to yield analog 217,an orange compound (77% yield).

Example 81. Synthesis of Analog 218: To a solution of Illudin M (1.00equivalent), epsilon-maleimidocaproic acid (1.33 equivalent), DMAP (0.18equivalent) in 1.0 mL of methylene chloride (CH₂Cl₂) at 0° C., is addedDCC (dicyclohexylcarbodiimide; 1.33 equivalent) in methylene chloride(CH₂Cl₂). The mixture is stirred for 2.25 hours, diluted with hexane andprecipitated is filtered. Residual solvent is evaporated off, and oilresidue is chromatographed on a silica column (2:1 hexane:ethyl acetate)to yield analog 218, an orange compound (83% yield).

Example 82. Synthesis of Analog 219: Analog 204 (33.4 mg) is dissolvedin 1.0 mL of anhydrous pyridine under a nitrogen atmosphere, then DMAP(5.1 mg) is added, followed by 4-fluorosulfonyl-benzoyl chloride (86.1mg). The mixture is stirred for 90 minutes at room temperature Themixture is diluted with ethyl acetate, washed once with saturated coppersulfate solution, washed twice with water, then dried over MgSO₄,concentrated then chromatographed (20% ethyl acetate:hexane) to giveanalog 219.

Example 83. Synthesis of Analog 221: Prepared from Analog 207 bycoupling with Mu-His-Ser-Ser-Lys(Fmoc)-Leu-Gln-OH in DIC/HOBt for 5minutes, then 5% piperidine/DMF for 1 minute. Followed by TFA quenchingto yield analog 221 at 21% yield.

Example 84. Synthesis of Analog 222: Illudin M (63 mg) is dissolved in1.0 mL of anhydrous pyridine under a nitrogen atmosphere, then DMAP (6.4mg) is added, followed by 4-fluorosulfonyl-benzoyl chloride (86 mg). Themixture is stirred for 35 minutes at room temperature thenchromatographed (20% ethyl acetate:hexane) to give analog (70.9 mg).

Example 85. Synthesis of Analog 223: The disulthydryl peptide CNGRC isfirst converted to a cyclic disulfide peptide by dissolving 355 mg in3.0 mL DMSO, adding 9 mL of water, allowing to sit overnight at roomtemperature, followed by water removal on a rotoevaporator then DMSOremoval under high vacuum. The TFA salt of analog 179 (14.5 mg) isdissolved in DMF (2.0 mL) and the CNGRC disulfide peptide added (19.0mg), 60 μL of DIPEA is added, followed by gradual addition of a solutionof Py-BOP (19.6 mg) and HOBt (8.9 mg) in DMF (2.0 mL) over 150 minutesat room temperature. The reaction is stopped by adding two drops of TFAand water. The mixture is applied to a reverse phase column and analog223 is eluted with acetonitrile:water (1:4).

Example 86. Synthesis of Analog 224: Acylfulvene (116 mg) is dissolvedin ethanol (4.0 mL) with stirring, hydroxylamine hydrochloride (84.2 mg)added, Sodium acetate (233 mg) added, then refluxed for 70 minutes at85° C. The ethanol is removed, then ethyl acetate (10 mL) added todissolve crude product, then water (10 mL) added, the organic layer iswashed with brine, dried over Na₂SO₄, concentrated then chromatographed(20% ethyl acetate:hexane) to give analog 224 (63.7 mg, 54% yield).

Example 87. Synthesis of Analog 225: Illudin S (439 mg) is dissolved inethanol (15 mL) with stirring, hydroxylamine hydrochloride (233 mg)added, sodium acetate (933 mg) added, then refluxed for 130 minutes at85° C. The solution is cooled to room temperature, filtered, ethanol isremoved, then ethyl acetate (30 mL) added to dissolve crude product,then water (30 mL) added, the organic layer is washed with brine, driedover Na₂SO₄, concentrated then chromatographed (30%->50%,acetone:hexane) to give analog 225 (372 mg, 80% yield).

Example 88. Synthesis of Analog 226: Irofulven (37.6 mg) is dissolvedwith stirring in CH₂Cl₂, elaidic acid (180 mg. 1.3 equivalents) added,DMAP (15 mg) added, cooled to 0° C., then DCC (180 μL) in CH₂Cl₂ (640μL) added. Reaction mixture stirred at 0° C. for 1 hour, then additionalDCC (120 μL) added, and stirred for 2 more hours. Mixturechromatographed (20% ethyl acetate:hexane) to give analog 226 as ayellow oil (50.5 mg, 48% yield).

Example 89. Synthesis of Analog 227: Analog 009 (87 mg) is dissolvedwith stirring in CH₂Cl₂, elaidic acid (108 mg) added, DMAP (15.4 mg)added, cooled to 0° C., then DCC (0.5 mL) in CH₂Cl₂ (1.5 mL) added.Reaction mixture stirred at 0° C. for 3 hours, then the mixture directlychromatographed (20% ethyl acetate:hexane) to give analog 227 as ayellow oil (105 mg, 61% yield).

Example 90. Synthesis of Analog 228: Illudin S (86 mg) is dissolved withstirring in CH₂Cl₂, elaidic acid (202 mg) added, DMAP (15.4 mg) added,cooled to 0° C., then DCC (1.0 mL) in CH₂Cl₂ (3.0 mL) added. Reactionmixture stirred at 0° C. for 3 hours, then the mixture directlychromatographed (20% ethyl acetate:hexane) to give analog 228 as ayellow oil (198 mg, 77% yield).

Example 91. Synthesis of Analog 229: The elaidic ester of0-diphenylphosphine phenol is first prepared by dissolving with stirringin 3.0 mL of CH₂Cl₂ the O-diphenylphosphine phenol (91.3 mg), elaidicacid (94.5 mg, 1 equivalent), DMAP (9.4 mg). The solution is cooled to0° C. then DDC (0.44 mL, 1.0 M in CH₂Cl₂) is added with stirring for 3.5hours. The precipitate is filtered off and discarded. The elaidic esteris chromatographed and concentrated to dryness then dissolved in THF(1.0 mL). Analog 195 (26.1 mg) is dissolved in THF (1.0 mL) and water(80 μL) added. The elaidic ester solution is slowly added to the analog195 solution with stirring, and reacted for 22 hours at roomtemperature. The mixture is directly chromatographed (30%acetone:hexane) to give analog 229 (22.2 mg, 47% yield).

Example 92. Synthesis of Analog 230: Analog 308 (22 mg) is dissolved inanhydrous CH₂Cl₂ (1.5 mL), diisopropylethylamine (20 μL) added, and themixture cooled to 0° C., then methylsulfonyl chloride added (15 μL),mixture stirred at 0° C. for 1 hour, and allowed to warm to roomtemperature while being stirred for an additional hour. The mixture ischromatographed (30% ethyl acetate in hexane) to yield analog 230 (35%yield).

Example 93. Synthesis of Analog 231: Analog 308 (16 mg) is dissolved inanhydrous CH₂Cl₂ (1.5 mL), diisopropylethylamine (20 μL) added, and themixture cooled to 0° C., then tosyl chloride added (18.4 mg), mixturestirred at 0° C. for 1 hour, and allowed to warm to room temperaturewhile being stirred for an additional 3 hours. The mixture ischromatographed (30% ethyl acetate in hexane) to yield analog 231 (8.6mg).

Example 94. Synthesis of Analog 240: Analog 232 (25.1 mg) is dissolvedin anhydrous CH₂Cl₂ (2.0 mL), 154 of acetic anhydride added, and themixture cooled to room temperature, then DMAP added (5 mg), and stirredfor 25 minutes. The mixture is partially concentrated thenchromatographed (30% ethyl acetate in hexane) to yield analog 240 (26.6mg, 93% yield).

Example 95. Synthesis of Analog 254: Analog 009 (51.4 mg),4-carboxybenzene sulfonamide (59.4 mg), and DCC (39.6 mg) were dissolvedin anhydrous DMF (1.0 mL) at room temperature, stirred, then DMAP (15mg) added. The mixture was stirred for 2 hours at room temperature thensolid material was filtered off. The mixture was then chromatographed(1:1 ethyl acetate:hexane) to give analog 254 (38.6 mg, 45% yield).

Example 96. Synthesis of Analog 255: Analog 009 (244.3 mg) and sulfamoylchloride (157 mg) were dissolved in anhydrous DMAP (2.0 mL) at roomtemperature, and stirred for 3.5 hours. The mixture was concentratedunder high vacuum then chromatographed (30% ethyl acetate in hexane) togive analog 255.

Example 97. Synthesis of Analog 259: Analog 255 (64.7 mg),(diacetoxyiodo)benzene (64.7 mg), dirhodiumtetraacetate or Rh₂(OAc)₄ andmagnesium (16.8) dissolved in 5.0 mL of CH₂Cl₂ are heated to 70° C. andstirred for 7 hours. The mixture is filtered, concentrated, thenchromatographed (1:1 ethyl acetate:hexane) to give analog 259.

Example 98. Synthesis of Analog 262 and 263 (prepared together): Analog25 (44.7 mg) is dissolved in methanol (1.0 mL), Oxone® reagent (246 mg,3 equivalents) is dissolved in water (1.0 mL). The oxone solution isslowly added to the methanol solution with stirring at room temperaturefor 3.5 hours, then an additional amount of Oxone reagent added followedby stirring for 1.5 hours. Then 2 mL of saturated sodium sulfitesolution was added, followed by ethyl acetate extraction, dried overNa₂SO₄, concentrated then chromatographed (1:1 Ethyl acetate:hexane) toyield first analog 263 (21.4 mg) and then analog 262 (14.3 mg).

Example 99. Synthesis of Analog 284 and 289 (prepared together): Analog34 (174 mg) and uracil (227 mg) are dissolved in CH₂Cl₂ with stirringand the mixture cooled to 0° C. Then SnCL₄ (148.8 μL) is slowly added.The mixture is stirred at 0° C. for 80 minutes, then concentrated,chromatographed (2->5% methanol:CH₂Cl₂) to give analog 284 (68.9 mg, 33%yield) and analog 289 (21.6 mg, 10% yield).

Example 100. Synthesis of Analog 285: Analog 34 (25 mg) is dissolved inethanol, and O-(tert-Butyldimethylsilyl) hydroxylamine (25 mg) is addedfollowed by stirring for 2 hours at room temperature. The secondaryamine intermediate (9 mg) is recovered by chromatography (30% ethylacetate:hexane), dissolved in CH₂Cl₂, and reacted with sulfamoilchloride (ClSO₂NH₂, 5 mg) and DABCO (2 mg) with stirring for one hour,then additional sulfamoil chloride (6 mg) was added with stirring foranother 1.5 hours. The TPS blocked product was recovered bychromatography (30% ethyl acetate:hexane), and the TPS group was removedin THF by adding TBAF (Tetra-n-butylammonium fluoride). The TPS groupcan also be removed by dissolving the TPS product in pyridine and THF at0° C., then adding HF-pyridine overnight. After TPS deblocking themixture is chromatographed (50% ethyl acetate:hexane) to give analog285.

Example 101. Synthesis of Analog 286 and 287 (prepared together): Theketone groups on 5-fluorouracil are first blocked with TMS groups bydissolving 5-fluorouracil (610 mg) and (NH₄)₂SO₄ in HMDS (10 mL) under anitrogen atmosphere. The solution is refluxed at 142° C. for 2.5 hours,cooled to 60° C. and excess HMDS distilled off, then concentrated todryness under high vacuum. Analog 34 (180 mg) and the di-TMS5-fluorouracil are dissolved in CH₂Cl₂ (5.0 mL) with stirring and themixture cooled to 0° C. Then SnCL₄ (120 μL) is slowly added drop wise.The mixture is stirred at 0° C. for 3.5 hours, then concentrated,chromatographed (80% ethyl acetate:hexane) to give analog 286 (18.9 mg,9% yield) and analog 287 (84 mg, 38% yield).

Example 102. Synthesis of Analog 289: See the preparation of analog 284for the preparation of analog 289 (284 and 289 prepared simultaneouslythen separated by chromatography).

Example 103. Analogs 299 and 300 (prepared together): Analogs 299 and300 are prepared in equal amounts from Illudin S using the Mitsunobureaction. Illudin S is directly reacted with HN₃ (PPh3, DEAD, benzene)at 0° C. under nitrogen for 45 minutes. Mitsunobu, O. Synthesis 1:1-28,1981.

Example 104. Synthesis of Analog 301: Irofulven (31.6 mg, 0.128 mmol),5-benzoylvaleric acid (35.8 mg, 0.174 mmol) and DMAP (4.7 mg) isdissolved in CH₂Cl₂ (2 mL) under a nitrogen atmosphere, cooled to 0° C.,the DCC added (170 μL of 1.0M solution in CH₂Cl₂). The mixture isstirred for 60 minutes then diluted with hexane (10 mL) and filtered.The organic layer is further diluted with CH₂Cl₂, washed with water,then saturated NaHCO₃ then brine, dried with MgSO₄, concentrated, thendissolved in CH₂Cl₂, filtered and chromatographed (10:3 hexane:ethylacetate), appropriate fractions collected, pooled, concentrated thenchromatographed (3:1 hexane:ethyl acetate) to give analog 301 (23.2 mg,42% yield).

Example 105. Analogs 302 and 303 (prepared together): Illudin S (100 mg,0.378 mmol) is benzoylated by dissolving in pyridine (1.0 mL) thenadding 3,5-dintirobenzoyl chloride (110 mg, 0.5 mmol) at roomtemperature and stirring for 24 hours. The mixture is poured ontocrushed ice then extracted with CH₂Cl₂ (10 mL), which is washed twicewith water (20 mL). The organic layer is dried over Na₂SO₄ andconcentrated to yield analogs 302 and 303. The two analogs can beseparated by column chromatography (1:1 hexane:ethyl acetate).

Example 106. Synthesis of Analog 304: Analog 009 (84.6 mg) is dissolvedin anhydrous CH₂Cl₂ (3.0 mL), DCC added (81.2 mg), mixture cooled to 0°C., propiolic acid (35 μL) added, then the reaction started with DMAP(15 mg), stirred and allowed to warm to room temperature over 1 hour.The mixture was filtered to remove solids then chromatographed (30%ethyl acetate in hexane) to give analog 304 (60% yield).

Example 107. Synthesis of Analog 305: Analog 009 (99.1 mg) is dissolvedin anhydrous CH₂Cl₂ (3.0 mL), pyridine (150 μL) added, thenβ-nitrophenylchloroformate and stirred for 3.5 hours at roomtemperature. The mixture was concentrated, hexane (20 mL) added, andprecipitate filtered before chromatographing (50% ethyl acetate inhexane) to give analog 305 (50% yield).

Example 108. Synthesis of Analog 306: Analog 009 (244 mg) is dissolvedin anhydrous CH₂Cl₂ (4.0 mL), tosyl chloride (181 mg) added, the mixturecooled to 0° C., to which an aliquot of pyridine (80 μL) is added. Themixture stirred at 0° C. for 1 hour, and allowed to warm to roomtemperature while being stirred for an additional 20 hours. The mixtureis concentrated then chromatographed (50% ethyl acetate in hexane) toyield analog 306.

Example 109. Synthesis of Analog 307: A solution of 1.0 M N₃H in benzeneis first prepared by mixing 654 mg N₃H, 0.65 mL water, in 10 mL ofbenzene. The mixture is cooled to 0° C., 0.5 mL of concentrated H₂SO₄added, and allowed to warm slowly to room temperature and then stirredfor 80 minutes. Next PPh₃ (590 mg) is dissolved in anhydrous THF (1.5mL) and cooled to 0° C. Then 2.1 mL of N3H 1.0 M solution is added,followed by DEAD (0.475 mL) then Illudin S (282 mg in 1.0 mL anhydrousTHF). The mixture is stirred for 3 hours at 0° C., warmed, concentrated,followed by chromatography (30% ethyl acetate in hexane) to give analog307.

Example 110. Synthesis of Analog 308: Analog 307 (100 mg) is dissolvedin anhydrous THF (3.0 mL) at room temperature and PPH3 added (306 mg, 3equivalents). The mixture is stirred for 5 hours at room temperature,then the reaction stooped by adding water (0.15 mL). The mixture isheated to 85° C. for 30 minutes, then concentrated and chromatographed(20% methanol in ethyl acetate) to give analog 308.

Example 111. Synthesis of Analog 309: Analog 204 was reacted with HN₃(DEAD, THF) to yield the azide analog 309 at 68% yield.

Example 112. Synthesis of Analog 310: Irofulven (42.9 mg),4-carboxybenzene sulfonamide (41.4 mg), and DCC (38.4 mg) were dissolvedin anhydrous DMF (1.0 mL) at room temperature, stirred and then DMAP (10mg) added. The mixture was stirred for 75 minutes at room temperaturethen solid material was filtered off. The mixture was thenchromatographed (1:1 ethyl acetate:hexane) to give analog 310 (40%yield).

Example 113. Synthesis of Analog 311: Illudin M (32.4 mg),4-carboxybenzene sulfonamide (39.7 mg), and DCC (24.4 mg) were dissolvedin anhydrous DMF (1.0 mL) at room temperature, stirred, then DMAP (15mg) added. The mixture was stirred for 75 minutes at room temperature,allowed to warm to room temperature, then stirred for 22 hours. Thesolid material was filtered off and the mixture was then chromatographed(1:1 ethyl acetate:hexane) to give analog 311 (35% yield).

Example 114. Synthesis of Analog 312: Irofulven (1.18 grams) isdissolved in anhydrous CH₂Cl₂ (4.0 mL), tosyl chloride (1.1 equivalent)added, the mixture cooled to 0° C., then pyridine (0.4 mL) added. Themixture stirred at 0° C. for 1 hour, and allowed to warm to roomtemperature while being stirred for an additional 3 hours. The mixtureis concentrated then chromatographed (50% ethyl acetate in hexane) toyield analog 312.

Example 115. Synthesis of Analog 313: Analog 308 (31 mg) is dissolved inanhydrous CH₂Cl₂, cooled to 0° C., with stirring thendiisopropylethylamine added (45 μL), then fluorophenylsulfonyl chlorideadded (36 μL) for 3 hours at 0° C. Mixture is directly chromatographed(20% ethyl acetate:hexane) to give analog 313 (23.3 mg).

Example 116. Synthesis of Analog 314: Analog 009 is dissolved inanhydrous CH₂Cl₂ (4.0 mL), tosyl chloride (1.1 equivalent) added, themixture cooled to 0° C., then pyridine (0.4 mL) added. The mixturestirred at 0° C. for 1 hour, and allowed to warm to room temperaturewhile being stirred for an additional 3 hours. The mixture isconcentrated then chromatographed (50% ethyl acetate in hexane) to yieldanalog 314.

Example 117. Synthesis of Analog 315: Irofulven was dissolved in asolution of 2,5 dimethylpyrrole (4 fold excess molar solution) in 5 mLof dry CH₂Cl₂ at −78° C.. Boron trifluoride (equivalent molar amount tothe irofulven) was slowly added with stirring. The reaction was allowedto stir for 2 more hours at −78° C., then water slowly added. Themixture was extracted twice with 2 fold equivalent volumes of ethylacetate, the organic extracts combined, washed with saturated NaHCO₃,water, brine, then dried over MgSO₄. The solution was concentrated undervacuum until a red residue remained, which was chromatographed on silicagel (50% ethyl acetate in hexane) to yield analog 315 (30% yield).

Example 118. Synthesis of Analog 316: Analog 316 was prepared bydissolving Illudin S (20 mg) in pyridine (0.5 mL) and then4-fluorosulfonylbenzoly chloride (equivalent molar amount) was added tothe mixture in an ice bath. The solution is allowed to warm slowly andthen react overnight. The liquid was then removed under reduced pressureuntil a crude residue remained. Rather than recrystallize fromchloroform, the residue was instead chromatographed on a standard silicagel column using hexane-ethylacetate (1:1). The mono-adduct (analog316), a di-adduct and a small amount of unreacted Illudin S wererecovered in separate eluates.

Example 119. N₃H 1.0 M Solution: A solution of 1.0 M N₃H in benzene isfirst prepared by mixing 654 mg N₃H, 0.65 mL water, in 10 mL of benzene.The mixture is cooled to 0° C., 0.5 mL of concentrated H₂SO₄ added, andallowed to warm slowly to room temperature and then stirred for 80minutes.

Example 120. Synthesis of Analog 193: Irofulven (221 mg, 0.897 umol) isdissolved in anhydrous THF (1.5 mL), then PPh₃ (261 mg, 0.995 umol) isadded, then 1.0 M N₃H solution (1.0 mL, 1.0 mmol) under nitrogenatmosphere. The solution is cooled to −40° C., and then DIAD (0.21 mL,1.013 umol) added and stirred for 30 minutes at 0° C. then diluted withhexane, and filtered to remove precipitate. The mixture is concentratedthen chromatographed (30% ethyl acetate:hexane) to give analog 193 (171mg, 71%).

Example 121. Synthesis of Analog 195: Analog 009 (31.9 mg, 116 umol) isdissolved in anhydrous THF (3.0 mL), then PPh₃ (33 mg, 126 umol) isadded, then 1.0 M N3H solution (0.3061 mL) under nitrogen atmosphere.The solution is cooled to 0° C., DIAD (30 μL, 145 umol) added andstirred for 30 minutes at 0° C. then water (5 μL) is added to destroythe PPh₃. The mixture is concentrated then chromatographed (30% ethylacetate:hexane) to give analog 195 (24.9 mg, 72%).

The foregoing description of embodiments of the methods, systems, andcomponents of the present invention has been provided for the purposesof illustration and description. It is not intended to be exhaustive orto limit the invention to the precise forms disclosed. Manymodifications and variations will be apparent to one of ordinary skillin the relevant arts. For example, steps performed in the embodiments ofthe invention disclosed can be performed in alternate orders, certainsteps can be omitted, and additional steps can be added. The embodimentswere chosen and described in order to best explain the principles of theinvention and its practical application, thereby enabling others skilledin the art to understand the invention for various embodiments and withvarious modifications that are suited to the particular usedcontemplated. Other embodiments are possible and are covered by theinvention. Such embodiments will be apparent to persons skilled in therelevant art(s) based on the teachings contained herein. The breadth andscope of the present invention should not be limited by any of theabove-described exemplary embodiments, but should be defined only inaccordance with the following claims and their equivalents.

TABLE IA shows acylfulvene amine analogs which can be attached to abi-functional linker which can then be attached to a sulfhydryl reactinggroup of the AM using the reagent. Amine analog Reagent 97 121, 176, 2IT[2-iminothiolane (generated from) methyl 4-mercaptobutyrimidate], AMAS179, 184, 203, [N-(α-maleimidoacetoxy)-succinimide ester], BMPA[N-β-malemidopropionic acid], 205, 206, 207, BMPS[N-β-malemidopropyloxy)succinimide ester], C6-SFB [C6-succinimidyl 211,220, 244, 4-formylbenzoate], Citiolone[N-acetylhomocysteinethiolactone], DST 245, 254, 255, [disuccinimidyltartrate], EMCH [N-(episilon-maleimidocaproic acid) hydrazide], 264,266, 267, EMCS [N-(episilon-maleimideocaproyloxy)succinimide ester],GMBS [N- 270, 276, 283, (gamma-maleimideobutyrloxy)succinimide ester],KMUA [N-kappa- 285, 294, 295, maleimidoundecanoic acid], KMUH[N-(kappa-maleimidoundecanoic acid) 296, 297, 308, hydrazide], LC-SMCC[succinimidyl 4-(N-maleimidomethyl)cyclohexane-1- 310, 311carboxy-(6-amidocaproate)], LC-SDPD [succinimidyl 6-(3′-(2-pyridyl-dithio)propionamido)hexanoate], MBS [m-maleimidobenzoyl-N-hydroxysuccinimide ester], MCP [methyl 3-mercaptopropionimidate], MPBH[4-(4-N-maleimidophenyl)-butyric acid hydrazide], M2C2H [4-(N-maleimidomethyl)cyclohexanee-1-1carboxyl-hydrazide], NPIA [p-nitrophenyliodoacetate], PDPH [3-(2-pyridyldithio)propionyl hydrazide], PDTP [3-2(pyridyldithio)propionate], PMPI [N-(p-maleimidophenyl)isocyanate],SATA [succinimidyl S-acetylthioacetate], SATP [succinimidylacetylthiopropionate], SFB [succinimidyl p-formylbenzoate], SFPA[succinimidyl p- formylphneoxyacetate], SHTH [succinimidyl4-hydrazidoterephthalate], SIAB[N-succinimidyl(4-iodoacetyl)-aminobenzoate], SIAC [succinimidyl 4-(((iodoacetyl)amino)methyl)-cyclohexane-1-caroxylate], SIACX[succinimidyl 6- ((((4(iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)aminohexanoate], SIAX [succinimidyl6-((iodoacetyl)amino)hexanoate], SIAXX [succinimidyl 6-(6-(((iodoacetyl)amino)-hexanoyl)aminohexanoate], SAMSA [S-acetylmercaptosuccinic anhydride], SMCC [succinimidyl4-(N-maleimidomethyl)- cyclohexane-1-carboxylate], SM(PEG)2[NHS-PEO2-maleimide or maleimide PEG2 N-hydroxysuccinimide], SM(PEG)4[NHS-PEO4-maleimide or maleimide PEG4 N-hydroxysuccinimide], SM(PEG)8[NHS-PEO8-maleimide or maleimide PEG6 N-hydroxysuccinimide], SM(PEG)12[NHS-PEO12-mleimide or maleimide PEG8 N-hydroxysuccinimide], SMPB[succinimidyl 4-(para-maleimido- phenyl)butyrate], SMPH[succinimidyl-6-(beta- maleimidopropionamido)hexanoate], SMPT[4-succinimidyloxycarbonyl-methyl- alpha-(2-pyridyldithio)toluene], SPDP[N-succinimidyl 3-(2- pyridyldithio)propionate], Sulfo-DST[sulfo-disuccinimidyl tartrate], Sulfo- EMCSN-(episilon-maleimidocaproyloxy)sulfosuccinimide], Sulfo-GMBS [N-(gamma-maleimidobutyrloxy)sulfosuccinimide ester], Sulfo-KMUS [N-(kappa-maleimidoundecanoyloxy)sulfosuccinimide ester], Sulfo-LC-SMPT[sulfosuccinimidyl6-(alpha-methyl-alpha-(2-[pyridyldithio)-toluamido)hexanoate],Sulfo-LC-SPDP [sulfosuccinimidyl 6-(3′-(2-pyridyl-dithio)propionamido)hexanoate], Sulfo-MBS [m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester], Sulfo-SIAB[sulfo-succinimidyl(4-iodoacetyl)- aminobenzoate], Sulfo-SMCC[sulfosuccimidyl 4-(N-maleimido- methyl)cyclohexane-1-carboxylate],Sulfo-SMPB [sulfosuccimidyl 4-(p- maleimidophenyl)butyrate]

TABLE IB Acylfulvene amine analogs attached to a linker which isattached to a photoactivatable group at the other terminus. Amine analogReagent 97, 121, 176, ANB-NOS [N-5-azido-2-nitrobenzyloxy-succinimide],NHS-ASA [N- 179, 184, 203, hydroxysuccinimidyl-4-azidosalicylic acid],SADPH [N-succinimidyl (4′- 205, 206, 207,azidophenyl)1,3′-dithiopropionate], SANPAH [N-succinimidyl 6-(4′azido-211, 220, 244, 2′-nitrophenylamino)hexanoate], SPB[succinimidyl-(4-psoralen- 245, 254, 255, 8y;oxy)butyrate], Sulfo-HSAB[N-hydroxysulfosuccinimidyl-4- 264, 266, 267, azidobenzoate],Sulfo-NHS-LC-ASA [sulfosuccinimidyl(4-azido- 270, 276, 283,salicylamido)hexanoate], Sulfo-SADP [sulfosuccinimidyl(4-azido- 285,294, 295, phenyldithio)propionate], Sulfo-SAED [sulfosuccinimidyl2-(7-azido-4- 296, 297, 308,methylcoumarin-3-acetamido)ethyl-1,3′-dithiopropionate], Sulfo-SASD 310,311 [sulfosuccinimidyl 2-(p-azido-salicylamido)ethyl1,3′-dithiopropionate], Sulfo- SFAD[sulfosuccinimidyl(perfluoroazidobenzamido)ethyl 1,3′-dithiopropionate], Sulfo-SAND [sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl 1,3′-dithiopropionate], Sulfo-SANPAH[sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino)hexanoate]

TABLE IC Acylfulvene amine analogs attached to a linker which isattached to an amine reactive group at the other terminus. Amine analogReagent 97, 121, 176, BS2G-do [bis(sulfosuccinimidyl)glutarate-d0],BS2G-d4 [bis 179, 184, 203, (sulfosuccinimidyl)2,2,4,4,glutarate-d4],BS3 (or Sulfo-DSS) [bis 205, 206, 207, (sulfosuccinimidyl)suberate],BS3do [bis(sulfosuccinimidyl)suberate], 211, 220, 244, BS3d4[bis(sulfosuccinimidyl)2,2,7,7-suberate-d4], BS(PEG)5 [bis(NHS) 245,254, 255, PEO5], BSOCOES[bis(2-(succininidoxycarbonyloxy)ethyl)sulfone], 264, 266, 267, DMA[dimethyl adipimidate], DMP [dimethyl pimelimidate], DMS 270, 276, 283,[dimethyl suberimidate], DFDNB [1,5,-difluoro-2,4-dinitrobenzene], 285,294, 295, DFDNPS [4,4′-difluoro-3,3′-dinitrophenylsulfone], DSG[disuccinimidyl 296, 297, 308, glutarate], DSS [disuccinimidylsuberate], DST [disuccinimidyl tartarate], 310, 311 DSP or Lomant'sreagent [dithiobis(succimidylpropionate)], DTBP dimethyl3,3′-dithiobispropionimidate], DTSSP (sulfo-DSP) = [3,3′-dithio-bis(sulfosuccinimidylpropionate)], EGS [ethylene glycol bis(succinimidylsuccinate)], PMPI [N-(4-Isocyanatophenyl)maleimide],Sulfo-EGS [ethylene glycol bis(sulfo-succinimidyl-succinate)]

TABLE ID Acylfulvene amine analogs attached to a linker which isattached to a reactive group capable of reacting with an aldehyde,carbonyl or carboxylate group at the other terminus. Amine analogReagent 97, 121, 176, 179, C6-SANH [C6-succinimidyl 4-hydraznonicotinateacetone hydrazone] 184, 203, 205, 206, SANH [succinimidyl4-hydraznonicotinate acetone hydrazone] 207, 211, 220, 244, EDC[1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride] 245, 254,255, 264, 266, 267, 270, 276, 283, 285, 294, 295, 296, 297, 308, 310,311

TABLE IIA shows acylfulvene carboxylate analogs which can be attached toa bi-functional linker which can be attached to a sulfhydryl reactinggroup of the AM. Carboxylate analog Reagent 29, 37, 38, 64, 97, 98, BMPH[N-β-maleimidopropionic acid) hydrazide-trifluoroacetic 106, 117, 118,145, acid salt], EMCH [N-(episilon-maleimidocaproic acid) hydrazide]160, 162, 177, 178, KMUH [N-(kappa-maleimidoundecanoic acid)hydrazide],MPBH 181, 258 [4-(4-N-maleimidophenyl)-butyric acid hydrazide], PDPH[3-(2- pyridyldithio)propionylhydrazide], SHTH [succinimidyl 4-hydrazidoterephthalate], M2C2H [4-(N-maleimidomethyl)cyclohexanee-1-1carboxyl-hydrazide], PMPI [N-(4-Isocyanato-phenyl)maleimide], AMBH [2-acetamido-4-mercaptobutyric acid hydrazide]

TABLE IIB shows acylfulvene carboxylate analogs which can be attached toa bi-functional linker, where the linker also contains aphotoactivatable reactive group which can attach to the AM. Carboxylateanalog Reagent 29, 37, 38, 64, 97, 98, ABH [p-azidobenzoyl hydrazide]106, 117, 118, 145, 160, ASBA [4-(p-azidosalicylamido)-butylamine] 162,177, 178, 181, 258

TABLE IIC shows acylfulvene carboxylate analogs which can be attached toa bi-functional linker, where the linker also contains an amino reactivegroup which can attach to the AM. Carboxylate analog Reagent 29, 37, 38,64, 97, EDC [1-ethyl-3-(3-dimethylamino- 98, 106, 117, 118, propyl)carbodiimide hydrochloride 145, 160, 162, 177, CMC [1-cyclohexyl-3-2(2-178, 181, 258 morpholinoethyl)carbodiimide] AADH [adipic aciddihydrazide] Woodward's Reagent K [N-ethyl-3- phen ylisoxazolium-3′sulfonate]

TABLE IID Acylfulvene carboxylate analog attached through carboxylategroup to a linker where the linker also contains an azlactone reactivegroup to attach to the AM. Carboxylate analog Reagent 29, 37, 38, 64,97, glycine or either an L or D amino acid including alanine, serine,threonine, 98, 106, 117, 118, cysteine, valine, leucine, isoleucine,methionine, proline, phenylalanine, 145, 160, 162, 177, tyrosine,tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, 178,181, 258 histidine, lysine, arginine, or nonstandard amino acidsincluding homo- cysteine, selenocysteine, pyrrolysine, carnitine,hypusine, lanthionine, 2-aminoisobutyric acid, dehydroalanine,gamma-aminobutyric acid, ornithine, citrulline, α-Amino-n-butyric acid,Norvaline, Norleucine, Pipecolic acid, Alloisoleucine,α,β-diaminopropionic acid, α,γ- diaminobutyric acid, Allothreonine,α-Amino-n-heptanoic acid, Homoserine, β-Amino-n-butyric acid,β-Aminoisobutyric acid, γ-Aminobutyric acid, isovaline, Sarcosine,N-ethyl glycine, N-propyl glycine, N-isopropyl glycine, N-methylalanine, N-ethyl alanine, N-methyl β-alanine, N-ethyl β-alanine,Isoserine, α-hydroxy-γ-amino- butyric acid, diaminopimelic acid,cystathione, aminoisobutyric acid, dehydroalanine, delta-aminolevulinicacid, 4-aminobenzoic acid, Hydroxyproline, Formylmethioinine,lanthionine, djenkolic acid, Pyroglutamic acid, Hypusine,carboxyglutamic acid, penicillamine, thialysine, quisqualic acid,canavine, azeticline-2-carboxylic acid, 2- dimethylglycine.

TABLE IIIA shows acylfulvene carbonyl analogs which can be attached to abi-functional linker which can be attached to a sulfhydryl reactinggroup of the AM using the reagent. Carbonyl analog Reagent 13, 27, 28,51, 83, 84, AMBH [2-acetamido-4-mercaptobutyric acid hydrazide, BMPH [N-124, 131, 144, 167, 184, β-maleimidopropionic acid)hydrazide-trifluoroacetic acid salt], 201, 207, 232, 233, 234, EMCH[N-(episilon-maleimidocaproic acid) hydrazide], KMUH 235, 237, 238, 239,240, [N-(kappa-maleimidoundecanoic acid)hydrazide], MPBH [4-(4-N- 243,276, 277, 278, 279, maleimidophenyl)-butyric acid hydrazide], PDPH[3-(2- 280, 281, 282, 286, 287, pyridyldithio)propionylhydrazide], SHTH[succinimidyl 4- 288, 289, 294, 295, 296, hydrazidoterephthalate] 297,298, 301, 302, 303,

TABLE IIIB shows acylfulvene carbonyl analogs which can be attached to abi-functional linker, where linker also contains photoactivatablereactive group which can attach to AM using reagent. Carbonyl analogReagent 13, 27, 28, 51, 83, 84, 124, 131, 144, 167, ABH [p-azidobenzoylhydrazide] 184, 201, 207, 232, 233, 234, 235, 237, 238, ASBA[4-(p-azidosalicylamido)-butylamine] 239, 240, 243, 276, 277, 278, 279,280, 281, 282, 286, 287, 288, 289, 294, 295, 296, 297, 298, 301, 302,303

TABLE IIIC shows acylfulvene carbonyl analog which can be attached to abi-functional linker, where linker also contains an amine reactive groupwhich can attach to the AM using the reagent. Carbonyl analog Reagent13, 27, 28, 51, 83, 84, 124, EDC [1-ethyl-3-(3-dimethylaminopropyl) 131,144, 167, 184, 201, 207, carbodiimide hydrochloride 232, 233, 234, 235,237, 238, CMC [1-cyclohexyl-3-2(2- 239, 240, 243, 276, 277, 278,morpholinoethyl)carbodiimide] 279, 280, 281, 282, 286, 287, C6-SANH[C6-succinimidyl 4- 288, 289, 294, 295, 296, 297, hydraznonicotinateacetone hydrazone] 298, 301, 302, 303 SANH [succinimidyl 4-hydraznonicotinate acetone hydrazone]

TABLE IVA shows acylfulvene aldehyde analogs which can be attached to abi-functional linker which can be attached to a sulfhydryl reactinggroup of the AM using the reagent. Aldehyde analog Reagent 8, 10, 11,13, BMPH [N-β-maleimidopropionic acid) hydrazide-trifluoroacetic acidsalt], 41, 144, 156, EMCH [N-(episilon-maleimidocaproic acid)hydrazide], KMUH [N-(kappa- 201 maleimidoundecanoic acid)hydrazide],MPBH [4-(4-N-maleimidophenyl)- butyric acid hydrazide], PDPH[3-(2-pyridyldithio)propionylhydrazide], SHTH [succinimidyl4-hydrazidoterephthalate], AMBH [2-acetamido-4- mercaptobutyric acidhydrazide], PMPI [N-(4-Isocyanatophenyl)maleimide], AMBH[2-acetamido-4-mercaptobutyric acid hydrazide]

TABLE IVB shows acylfulvene aldehyde analogs which can be attached to abi- functional linker where the linker also contains a photoactivatablereactive group which can attach to the AM using the reagent. Aldehydeanalog Reagent 8, 10, 11, 13, 41, ABH [p-azidobenzoyl hydrazide] 144,156, 201 ASBA [4-(p-azidosalicylamido)-butylamine]

TABLE IVC shows acylfulvene aldehyde analogs which can be attached to abi-functional linker where the linker also contains an amine reactivegroup which can attach to the AM using the reagent. Aldehyde analogReagent 8, 10, 11, 13, EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride] 41, 144, 156, 201 CMC[1-cyclohexyl-3-2(2-morpholinoethyl)carbodiimide] AADH [adipic aciddihydrazide] C6-SANH [C6-succinimidyl 4-hydraznonicotinate acetonehydrazone] SANH [succinimidyl 4-hydraznonicotinate acetone hydrazone]Carbohydrazide [1,3-diamonourea]

TABLE VA shows acylfulvene alcohol analogs which can be attached to abi-functional linker which can be attached to a sulfhydryl reactinggroup of the AM using the reagent. Alcohol analog Reagent Illudin S,Illudin M, 2, 6, 9, 15,19, 22, 23, 32, PMPI[N-(p-maleimidophenyl)isocyanate] 42, 56, 62, 63, 77, 78, 81, 90, 99,103, 117, 118, 119, 127, 128, 135, 136, 145, 155, 159, 162, 187, 200,204, 208, 277 & 279 & 280, 299, 300, 307, 308

TABLE VB shows acylfulvene alcohol analogs which can be attached to abi-functional linker, where the linker also contains an amine reactivegroup which can attach to the AM using the reagent. Alcohol analogReagent Illudin S, Illudin M, 2, 6, 9, 15,19, 22, 23, 32, CDI[N,N′-carbonyldiimidazole], DSC 42, 56, 62, 63, 77, 78, 81, 90, 99, 103,117, [N,N′-disuccinimidyl carbonate], HSC [N- 118, 119, 127, 128, 135,136, 145, 155, 159, hydroxysuccinimidyl chloroformate] 162, 187, 200,204, 208, 277 & 279 & 280, 299, 300, 307, 308

TABLE VIA shows acylfulvene sulfhydryl analogs which can be attached toa bi-functional linker, where the linker also contains an amine reactivegroup which can attach to the AM using the reagent. Sulfhydryl analogReagent Analog 51 AMAS [N-(α-maleimidoacetoxy)-succinimide ester [BMPA[N-β- Terminal malemidopropionic acid] cysteine or n- BMPS[N-β-(malemidopropyloxy)succinimide ester] acetyl cysteine EMCH[N-(episilon-maleimidocaproic acid) hydrazide] EMCS[N-(episilon-maleimideocaproyloxy)succinimide ester] GMBS[N-(gamma-maleimideobutyrloxy)succinimide ester] KMUA[N-kappa-maleimidoundecanoic acid] KMUH [N-(kappa-maleimidoundecanoicacid) hydrazide] LC-SMCC [sucinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxy- (6-amidocaproate)] LC-SDPD[succinimidyl 6-(3′-(2-pyridyl-dithio)propionamido)hexanoate] MBS[m-maleimidobenzoyl-N-hydroxysuccinimide ester] M2C2H[4-(N-maleimidomethyl)cyclohexanee-1-1carboxyl-hydrazide] MPBH[4-(4-N-maleimidophenyl)-butyric acid hydrazide] PDPH[3-(2-pyridyldithio)propionylhydrazide] PMPI[N-(p-maleimidophenyl)isocyanate] SBAP [succinimidyl3-bromoacetamido)propionate] SHTH [succinimidyl4-hydrazidoterephthalate] SIA [N-succinimidyl iodacetate] SIAB[N-succinimidyl(4-iodacetyl)aminobenzoate] SMCC [succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate] SMPB [succinimidyl4-(para-maleimido-phenyl)butyrate] SMPH[succinimidyl-6-(beta-maleimidopropionamido)hexanoate] SM(PEG)2[NHS-PEO₂-maleimide or maleimide PEG2 N- hydroxysuccinimide] SM(PEG)4[NHS-PEO₄-maleimide or maleimide PEG4 N- hydroxysuccinimide] SM(PEG)8[NHS-PEO₈-maleimide or maleimide PEG6 N- hydroxysuccinimide] SM(PEG)12[NHS-PEO₁₂-mleimide or maleimide PEG8 N- hydroxysuccinimide] SMPH[succinimidyl-6-(beta-maleimidopropionamido)hexanoate] SMPT[4-succinimidyloxycarbonyl-methyl-alpha-(2-pyridyldithio)toluene] SPDP[N-succinimidyl 3-(2-pyridyldithio)propionate] Sulfo-EMCS[N-(episilon-maleimidocaproyloxy)sulfosuccinimide] Sulfo-GMBS[N-(gamma-maleimidobutyrloxy)sulfosuccinimide ester] Sulfo-KMUS[N-(kappa-maleimidoundecanoyloxy)sulfosuccinimide ester] Sulfo-LC-SMPT[sulfosuccinimidyl 6-(alpha-methyl-alpha-(2-[pyridyldithio)-toluamido)hexanoate], Sulfo-LC-SPDP [sulfosuccinimidyl6-(3′-(2-pyridyl-dithio)propionamido)hexanoate], Sulfo-MBS [m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester], Sulfo-SIAB[sulfosuccimidyl(4-iodo-acetyl)aminobenzoatel, Sulfo-SMCC[sulfosuccimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate],Sulfo-SMPB [sulfosuccimidyl 4-(p-maleimidophenyl)butyrate]

TABLE VIB shows acylfulvene sulfhydryl analogs which can be attached toa bi-functional linker, where the linker also contains a sulfhydrylreacting group which can attach to AM using reagent. Sulfhydryl analogReagent Analog 51, BMB [1,4-bis-maleimidobutane], Terminal BMDB[1,4-bis-maleimidyl-2,3- cysteine or hydroxybutyrate], BMH[bis-maleimidehexane], n-acetyl BMOE [bis-maleimideethanol], cysteineBM[PEO]2 [1, 8-bis-malemidodiethyene- glycol], BM[PEO]3 [1, 11-bis-malemidotriethyene-glycol], DPDPB [1,4-di(3′-(2′pyridyldithio)propionamido)butane], DTME [dithio-bis-(sulfosuccinimidylpropionate)], HBVS [1,6-hexane-bis-vinylsulfone]

TABLE VIC shows acylfulvene sulfhydryl analogs which can be attached toa bi-functional linker, where linker also contains photoactivatablereactive group which can attach to AM using reagent. Sulfhydryl analogReagent Analog 51,Terminal APDP [N-(4-(p-azidosalicylamido)butyl)-cysteine or acetyl cysteine 3′-(2′-pyridyldithio)propionamide]

TABLE VID shows acylfulvene sulfhydryl analogs which can be attached toa bi-functional linker, where the linker also contains a carboxylatereactive group which can attach to AM using reagent. Sulfhydryl analogReagent Analog 51,Terminal EMCA [N-(episilon-maleimidocaproic cysteineor n- acid)] acetyl cysteine

TABLE VII shows the cytotoxic data IC₅₀ values (micromolar, 2 hourexposure, N = 3, mean ± SD) for Illuclin M, analog 108 and analog 110for cells expressing the estrogen receptor (ER) (MCF7) and cells notexnressing the ER (HT29). Analog HT29 (ER Negative) MCF7 (ER positive)Illudin M 0.52 ± 0.10 0.48 ± 0.13 108 >55 14.1 ± 2.8  110 >19 2.0 ± 0.1

TABLE VIII shows the activity of PSA cleavable acylfulven analogs (210,215, 216, 221) and precursor analogs against PSA negative and PSApositive cell line (48 hour exposure, N = 3; mean ± SD; IC50 values innM). Prostate PC3 Prostate Prostate (negative DuPro LnCAP Analog PSA)(trace PSA) (positive PSA) Illudin S    16 ± 5       11 ± 3       15 ±3     204 (Illudin S tosylate) n.t. n.t. 3,300 ± 1,000 207(9-amine-leucine)   880 ± 330    450 ± 40      560 ± 60    211 (9-amine)  350 ± 80     280 ± 20      270 ± 50    212 (Illudin M-proline)   120 ±20      20 ± 2       120 ± 30    213 (Illudin S-tosylate- 2,200 ± 100   360 ± 80      900 ± 200   proline) 214 (Illudin S-Pro-   300 ± 50     90 ± 10      190 ± 30    Ser-Ser-HOAc) 0 (9-ester linkage/Ac- 4,700± 500  3,500 ± 400     810 ± 130   Hyp-Ser-Ser-Chg-G Gln-Ser-Pro) 215(Illudin S-tosylate n.t. n.t. > 20,000 ester/Ac-Hyp-Ser-SeChg-Gln-Gln-Ser-Pro) 16 (Illudin M/ester/Ac-   190 ± 10     280 ± 60     190 ± 30    Hyp-Ser-Ser-Chg-G Gln-Ser-Pro) 221 (211/amide >21,00013,000 ± 1,000   800 ± 100   or nonester) Mu-His-Ser-Ser- Lys(Fmoc)-Leu-Gln-Leu n.t. denotes not tested

TABLE IX showing peptides cleaved by proteases. Protease Peptide SEQ.ID's PSA His-Ser-Ser-Lys-Leu-Gln-X SEQ. ID. 104Mu-His-Ser-Ser-Lys-Leu-Gln-X Mu-His-Ser-Ser-Lys-Leu-Gln-Lys-X SEQ. ID.106 Mu-His-Ser-Ser-Lys-Leu-EDA-Lys-X SEQ. ID. 108Mc-His-Ser-Ser-Lys-Leu-Gln-X Mc-His-Ser-Ser-Lys-Leu-Gln-XHyp-Ala-Ser-Chg-Gln-Ser-X SEQ. ID. 111 Hyp-Ala-Ser-Chg-Gln-Ser-Leu-XSEQ. ID. 116 Mu-Hyp-Ala-Ser-Chg-Gln-Ser-XMu-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-X Mc-Hyp-Ala-Ser-Chg-Gln-Ser-XMc-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-X Hyp-Ser-Ser-Chg-Gln-Ser-Ser-Pro-X SEQ.ID. 127 Mu-Hyp-Ser-Ser-Chg-Gln-Ser-Ser-Pro-XMc-Hyp-Ser-Ser-Chg-Gln-Ser-Ser-Pro-X4-O-Ac-Hyp-Ser-Ser-Chg-G1n-Ser-Ser-Pro-X SEQ. ID. 131Arg-Arg-Ser-Ser-Tyr-Tyr-Ser-Gly-X SEQ. ID. 132Mu-Arg-Arg-Ser-Ser-Tyr-Tyr-Ser-Gly-XMc-Arg-Arg-Ser-Ser-Tyr-Tyr-Ser-Gly-X Mc-Ser-Ser-Lys-Tyr-Gln-Leu-X SEQ.ID. 136 Mu-Ser-Ser-Lys-Tyr-Gln-Leu-XN-glutaryl-Hyp-Ala-Ser-chGly-Gln-Ser-Leu SEQ. ID. 137Mu-N-glutaryl-Hyp-Ala-Ser-chGly-Gln-Ser- LeuMc-N-glutaryl-Hyp-Ala-Ser-chGly-Gln-Ser- Leu Caspase-3Asp-Glu-Val-Asp-Pro-X SEQ. ID. 138 Mu-Asp-Glu-Val-Asp-Pro-XMc-Asp-Glu-Val-Asp-Pro-X Lys-Gly-Ser-Gly-Asp-Val-Glu-Gly-X SEQ. ID. 139Mu-Lys-Gly-Ser-Gly-Asp-Val-Glu-Gly-XMc-Lys-Gly-Ser-Gly-Asp-Val-Glu-Gly-X Cathepsin PLE-X B Gly-Phe-Leu-Gly-XSEQ. ID. 141 Lys-Lys-Phe-D-Ala-X SEQ. ID. 142 D-Ala-Phe-Lys-Lys-X SEQ.ID. 144 Mc-Poly-L-glutamic acid-X Mc-Gly-Phe-Leu-Gly-X SEQ. ID. 145Mc-Lys-Lys-Phe-D-Ala-X Mc-D-Ala-Phe-Lys-Lys-X Mu-Poly-L-glutamic acid-XMu-Gly-Phe-Leu-Gly-X Mu-Lys-Lys-Phe-D-Ala-X Mu-D-Ala-Phe-Lys-Lys-XVal-Cit-X FAP Lys-Gln-Glu-Gln-Asn-Pro-Gly-Ser-Thr-X SEQ. ID. 146Mu-Lys-Gln-Glu-Gln-Asn-Pro-Gly-Ser-Thr-XMc-Lys-Gln-Glu-Gln-Asn-Pro-Gly-Ser-Thr-X KallikreinGly-Lys-Ala-Phe-Arg-Arg-X SEQ. ID. 171 2 Mu-Gly-Lys-Ala-Phe-Arg-Arg-XMc-Gly-Lys-Ala-Phe-Arg-Arg-X MMP-2/-9/ Glu-Pro-Cit-Gly-Hof-Tyr-Leu-XSEQ. ID. 172 Mu-Glu-Pro-Cit-Gly-Hof-Tyr-Leu-XMc-Glu-Pro-Cit-Gly-Hof-Tyr-Leu-X Gly-Ile-Leu-Gly-Val-Pro-X SEQ. ID. 173Mu-Gly-Ile-Leu-Gly-Val-Pro-X Mc-Gly-Ile-Leu-Gly-Val-Pro-XGly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-X SEQ. ID. 174Mu-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-XMc-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-X MMP-7Lys-Arg-Ala-Leu-Gly-Leu-Pro-Gly SEQ. ID. 175Mu-Lys-Arg-Ala-Leu-Gly-Leu-Pro-Gly Mc-Lys-Arg-Ala-Leu-Gly-Leu-Pro-GlyArg-Pro-Leu-Ala-Leu-Trp-Arg-Ser SEQ. ID. 176Mu-Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser Mc-Arg-Pro-Leu-Ala-Leu-Trp-Arg-SerTOP Ala-L-L-Ala-L-Ile Mu-Ala-L-L-Ala-L-Ile Mc-Ala-L-L-Ala-L-Ile uPAD-Ala-Phe-Lys or SEQ. ID. 177 D-Ala-Phe-Lys-PABC CathepsinGly-Gly-Pro-Nle-X SEQ. ID. 178 K Mu-Gly-Gly-Pro-Nle-XMc-Gly-Gly-Pro-Nle-X Plasmin D-Ala-Phe-Lys-Lys-X SEQ. ID. 179Mu-D-Ala-Phe-Lys-Lys-X Mc-D-Ala-Phe-Lys-Lys-X D-Ala-Phe-Lys-XMu-D-Ala-Phe-Lys-X Mc-D-Ala-Phe-Lys-X ThrombinPoly-L-Lys-Gly-D-Phe-Pip-Arg-Ser-Gly-Gly- SEQ. ID. 180 Gly-Gly-Gly-XTrypsin Poly-L-Lysine-Gly-Ala-Ser-D-Arg-Phe-Thr- SEQ. ID. 181 Gly-X

In Table IX, the letter ‘X’ denotes the end attached to the medicant,Chg denotes cyclohexyl glycine, Cit denotes citrulline, EDA denotesethanyl-D-Alanine, Hof denotes homophenylalanine, Hyp denotes4-hydroxyproline, Mc denotes morpholinocarbonyl (carboxy-terminalprotecting group), Mu denotes 4-morpholine-carbonyl (amino-terminalprotecting group), Nle denotes norleucine, PABC denotespara-aminobenzoylcarboxyl, PLE denotes Poly-L-glutamic acid, Pip denotespiperidine.

TABLE X shows different Linker Strategies. Linker Reactive Group* IDerFunctional Group Bond product FSB 1220 Carboxylate Ester FSB 1220Hydroxyl Ether Isothiocyanate 1241 Primary Amine Isothiourea Isocyanate1242 Primary Amine Isourea Cyanate ester Primary Amine Isourea AcylAzide 1243 Primary Amine Amide NHS Ester 1244 Primary Amine AmideSulfonyl chloride 1245 Primary Amine Sulfonamide Tosylate Ester ThiolThioether Tosylate Ester Primary Amine Secondary Amine Tosylate EsterHydroxyl Ether Tresyl Ester Primary Amine Sulfonamide Aldehyde PrimaryAmine Secondary Amine Epoxide Primary Amine Secondary Amine CarboxylatePrimary Amine Carbamate Aryl Halide (Like Primary Amine ArylamineFluorobenzene) Imidoester 1248 Primary Amine Amidine CarbodiimidesPrimary Amine Amide (eg EDC or CMC) Diimidazoles (like CDI) PrimaryAmine Carbamate Maleic anhydride Primary Amine Amide AlkylphosphatePrimary Amine Phosphoramidate Succinic anhydride 1247 Primary AmineAmide (like DSC) Fluorophenyl esters Primary Amine AmideN,N′-disuccinimidyl Primary Amine Carbamate carbonateN-hydroxylsuccinimidyl Primary Amine Carbamate chloroformate Haloalkyl(like Sulfhydryl Thioester Iodoacetyl) Maleimide (like NEM) SulfhydrylThioether MAL I 1210 Sulfhydryl Thioether MAL I 1211 SulfhydrylThioether Maleimide Hexadienes 2 + 4 Aziridine Sulfhydryl ThioetherAcryloyl Sulfhydryl Thioether Flurobenzene Sulfhydryl Aryl ThioetherPyridyl disulfide Sulfhydryl Disulfide bond 5-thio-2-nitrobenzoicSulfhydryl Disulfide bond acid (TNB) Vinylsulfone SulfhydrylBeta-thiosulfonyl (like HBVS) Diazoalkane or Carboxylate EsterDiazoacetate N,N′-carbonyl Hydroxyl Carbamate diimidazole IsocyanateHydroxyl Carbamate Haloacetyl or alkyl Hydroxyl Ether halide AminooxyAldehyde Oxime Hydroxylamine Aldehyde Oxime Photolysis Aryl AzideNucleophilic addition Photolysis Halogenated Nucleophilic Aryl Azidaddition Azide/copper catalyst Alkene Triazoline Azide/copper catalystAlkyne Triazole Aldehyde/NaCNBH3 Primary Amine Secondary Amine Aminoacid 1230 Carboxylate/DCC Azlactone Azlactone Primary Amine AmideWoodward′s/Carboxylate Primary Amine Amide DSP or DTSSP Primary AminesDisulfide DSS Primary Amines Amide DST and sulfo-DST Primary AminesAmide BSOCOES and Primary Amines Amide sulfo-BSOCOES EGS and sulfo-EGSPrimary Amines Amide DSG Primary Amines Amide DMA Primary AminesAmidines DMP Primary Amines Amidines DMS Primary Amines Amidines DTBPPrimary Amines Disulfide Difluorobenzene Primary Amines Aryl secondaryderivatives amines (DFDNB or DFDNPS) Epoxide Sulfhydryl ThioetherEpoxide Hydroxyl Ether Carbohydrazide Aldehyde Hydrazone- Hydrazine SPDPor Sulfo-SPDP or Primary Amine Amide LC-SDPDP or Sulfo- LC-SDPDP SPDP orSulfo-SPDP or Sulfhydryl Disulfide LC-SDPDP or Sulfo- LC-SDPDP SMPT orPrimary Amine Amide Sulfo-LC-SMPT SMPT or Sulfhydryl DisulfideSulfo-LC-SMPT SMCC or Sulfo-SMCC Primary Amine Amide or LC-SMCC or SulfoLC-SMCC SMCC or Sulfo-SMCC Sulfhydryl Disulfide or LC-SMCC or Sulfo-LC-SMCC MBS and sulfo-MBS Primary Amine Amide MBS and sulfo-MBSSulfhydryl Thioether SIA/B and sulfo-SIA/B Primary Amine Amide SIAB andsulfo-SIAB Sulfhydryl Thioether SIAC or SIACX or Primary Amine AmideSIAX or SIAXX SIAC or SIACX or Sulfhydryl Thioether SIAX or SIAXX GMBSand sulfo-GMBS Primary Amine Amide GMBS and sulfo-GMBS SulfhydrylThioether MPBH Sulfhydryl Thioether MPBH Carbonyl Amide/Hydrazone M2C2HSulfhydryl Thioether M2C2H Carbonyl Amide PDPH Sulfhydryl Disulfide PDPHCarbonyl Amide/Hydrazone NHS-ASA Primary Amine Photoreactive Aryl AzideSulfo-NHS-ASA Primary Amine Photoreactive Aryl Azide Sulfo-NHS-LC-ASAPrimary Amine Photoreactive Aryl Azide HSAB and Sulfo-HSAB Primary AminePhotoreactive Azide with Amide SANPAH and Primary Amine PhotoreactiveSulfo-SANPAH Azide with Amide ANB-NOS Primary Amine Photoreactive Azidewith Amide SAND and Sulfo-SAND Primary Amine Photoreactive Azide withAmide SADP and Sulfo-SADP Primary Amine Photoreactive Azide with AmideSAPB and Sulfo-SAPB Primary Amine Photoreactive Azide with Amide SAEDand Sulfo-SAED Primary Amine Photoreactive Azide with Amide Sulfo-SAMCAPrimary Amine Photoreactive Azide with Amide Sulfo-SASD Primary AminePhotoreactive Azide with Amide Sulfo-SFAD Primary Amine PhotoreactiveAzide with Amide pNDPD Primary Amine Photoreactive Azide with AmidePNP-DTP Primary Amine Photoreactive Diazo with Amide APDP SulfhydrylPhotoreactive Azide with Thioether ABH Aldehyde Photoreactive Azide withHydrazor ASBA Carboxylate Photoreactive Azide with Amide SPB PrimaryAmine Photoreactive Psoralen group with Amide PMPA or PMPS SulfyhydrylThioether SANH or SHNH Primary Amine Amide or SHTH SANH or SHNH AldehydeHydrazone or SHTH BMPA or BMPS Sulfhydryl Thioether BMPA or BMPS PrimaryAmine Amide SATA or SATP Primary Amine Amide or SAMSA SATA or SATPHydroxylamine Sulfhydryl or SAMSA AMBH Aldehyde Hydrazone PMPISulfhydryl Thioether PMPI Hydroyxl Carbamate AADH Aldehyde HydrazoneAMAS Primary Amine Amide AMAS Sulfhydryl Thioether KMUS or Sulfo-KMUSPrimary Amine Amide KMUS or Sulfo-KMUS Sulfhydryl Thioether EMCH or EMCSPrimary Amine Amide or sulfo-EMCS EMCH or EMCS Sulfhydryl Thioether orsulfo-EMCS BS2 or BS3 or Amine Amide BS(PEG)5 series Citiolone PrimaryAmine Amide with free Sulfhydryl SMPB or Sulfo-SMPB Primary Amine Amideor SMPH or SBAI SMPB or Sulfo-SMPB Sulfhydryl Thioether or SMPH or SBAIWoodward′s Reagent K Carboxylate Enol Ester Intermediate “ “ Enol EsterPrimary Amine Amide Intermediate KMUA Sulfhydryl Thioether KMUA maryAmine in Amide presenc EDC KMUH Sulfhydryl Thioether KMUH Aldehyde orHydrazone Carboxylate BMPH Sulfhydryl Thioether BMPH Aldehyde orHydrazone Carboxylate PDTP Primary Amine Amide SFB or SFPA Primary AmineAmide with free aldehyde SM(PEG)n Series Primary Amine Amide SM(PEG)nSeries Sulfhydryl Thioether DPDPB Two Sulfhydryls Two DisulfidesBMH[PEO]n series Two Sulfhydryls Two Thioethers BMH or BMOE TwoSulfhydryls Two Thioethers BMB or BMDB Two Sulfhydryls Two ThioethersDTME Two Sulfhydryls Two Thioethers with internal disulfide bond NPIAPrimary Amine Amide NPIA Sulfhydryl Thioether MCP Primary Amine AmidineAbbreviation in Table X have been defined previously in Tables I throughTable VI.

TABLE XI shows Illudin1 analogs. Analog IUPAC Name of IlludofulveneAnalog 1 106 5-(((3′S,6′R)-6′-hydroxy-2′,2′,4′,6′-tetramethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]-3′-yl)oxy)-5-oxopentanoic acid 2 107(3′S,6′R)-6′-hydroxy-2′,2′,4′,6′-tetramethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane- 1,5′-inden]-3′-yl((13S)-13-methyl-17-oxo- 7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl) glutarate 3 108(13S)-17-hydroxy-13-methyl- 7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl 3-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propanoate 4 109(13S)-17-hydroxy-13-methyl- 7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl ((3′S,6′R)-6′-hydroxy-2′,2′,4′,6′-tetramethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane- 1,5′-inden]-3′-yl) glutarate 5110 (13S)-13-methyl-17-oxo- 7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl 3-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]- 3′-yl)propanoate 6 111(10R,13S)-10,13-dimethyl-3-oxo- 2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren- 17-yl3-(6′-hydroxy-2′,4′-dimethyl- 7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propanoate 7 112(13S)-10,13-dimethyl-17-oxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propanoate 8 113(R)-3′-(but-3-en-1-yl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]- 7′(6′H)-one 9 114(6′R)-6′-hydroxy-2′,4′,6′-trimethyl-3′-(2-(oxiran-2-yl)ethyl)spiro[cyclopropane- 1,5′-inden]-7′(6′H)-one 10 115(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propanal oxime 11 116(R)-3′-(tert-butoxymethyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 12 1175-(((2′S,6′R)-3′-((4-carboxybutanoyl)oxy)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]-2′-yl)methoxy)-5-oxopentanoic acid 13 1185-(((2′S,6′R)-2′-(((3,5-dinitrobenzoyl)oxy)methyl)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]-3′-yl)oxy)-5-oxopentanoic acid 14 119(6′R)-3′-(3,4-dihydroxybutyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 15 120(R)-6′-hydroxy-3′-(3-((3-((S)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propylidene)hydrazineylidene)propyl)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 16 121(R)-2-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propylidene)hydrazine-1-carboxamide 17 122(R)-6′-hydroxy-2′,4′,6′-trimethyl-3′-(3-(2-phenylhydrazineylidene)propyl)spiro[cyclopropane-1,5′-inden]-7′(6′H)-one 18 123(R)-N′-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane- 1,5′-inden]-3′-yl)propylidene)-4-methylbenzenesulfonohydrazide 19 124(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propanal O-acetyloxime 20 125 (R)-3′-(3-(2-(2,4- dinitrophenyl)hydrazineylidene)propyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane- 1,5′-inden]-7′(6′H)-one21 126 (R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-carbaldehyde oxime 22127 2-hydroxy-4-((R)-6′-hydroxy- 2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)butanenitrile 23 128(6′R)-6′-hydroxy-3′-(3-hydroxybutyl)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 24 129(6′R)-2′,4′,6′-trimethyl-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-6′,7′-diol 25 130(R)-6′-hydroxy-3′-(3-(hydroxyamino)propyl)-2′,4′,6′-trimethylspiro[cyclopropane- 1,5′-inden]-7′(6′H)-one 26 131(R)-N-benzyl-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propanamide 27 133(E)-7-(chloromethylene)-5-hydroxy-5,9- dimethylspiro[3.5]non-8-en-6-one28 134 (E)-6-(chloromethylene)-4-hydroxy-4,8-dimethylspiro[2.5]oct-7-en-5-one 29 135((2′S,6′R)-3′,6′-dihydroxy-2′,4′,6′- trimethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′- inden]-2′-yl)methyl 4-nitrobenzoate30 136 ((2′S,6′R)-3′,6′-dihydroxy-2′,4′,6′-trimethyl-7′-oxo-2′,3′,6′,7′- tetrahydrospiro[cyclopropane-1,5′-inden]-2′-yl)methyl 4-(N- acetoxyacetamido)benzoate 31 137((2′S,6′R)-6′-hydroxy-2′,4′,6′- trimethyl-3′,7′-dioxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′- inden]-2′-yl)methyl 4-nitrobenzoate32 138 ((2′S,6′R)-6′-hydroxy-2′,4′,6′-trimethyl-3′,7′-dioxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]-2′-yl)methyl 4-(N- acetoxyacetamido)benzoate 33 139(2′S,6′R)-6′-hydroxy-2′,4′,6′-trimethyl-2′-(((4-nitrobenzoyl)oxy)methyl)-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane- 1,5′-inden]-3′-yl4-nitrobenzoate 34 140((2′S,6′R)-3′-((4-(N-acetoxyacetamido)benzoyl)oxy)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]-2′-yl)methyl 4-(N- acetoxyacetamido)benzoate 35 141 dimethyl(5′R)-4′,5′-dihydroxy-5′,7′,9′-trimethyl-4′,5′-dihydro-1′H-spiro[cyclopropane-1,6′-[1,3a]ethenoindene]-2′,3′-dicarboxylate 36 142 dimethyl(5′R)-5′-hydroxy-5′,7′,9′-trimethyl-4′-oxo-4′,5′-dihydro-1′H-spiro[cyclopropane-1,6′-[1,3a]ethenoindene]-2′,3′-dicarboxylate 37 143(R)-6′-hydroxy-1′,2′,4′,6′-tetramethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one 38 144(R)-2-((2′-ethyl-6′-hydroxy-4′,6′-dimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane- 1,5′-inden]-3′-yl)methoxy)ethylacetate 39 145 (R)-5-((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methoxy)-5-oxopentanoic acid 40 146(R)-4-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)butanenitrile 41 147(R)-3′-((benzo[d]thiazol-2-ylthio)methyl)- 6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one 42 148(R)-3′-((benzo[d]oxazol-2-ylthio)methyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane- 1,5′-inden]-7′(6′H)-one43 149 (R)-6′-hydroxy-2′,4′,6′-trimethyl-3′-(((1-methyl-1H-tetrazol-5-yl)thio)methyl)spiro[cyclopropane- 1,5′-inden]-7′(6′H)-one 44150 (R)-6′-hydroxy-2′,4′,6′-trimethyl-3′-(((5-methyl-1H-benzo[d]imidazol-2- yl)thio)methyl)spiro[cyclopropane-1,5′-inden]-7′(6′H)-one 45 151(R)-6′-hydroxy-2′,4′,6′-trimethyl-3′-(((1-phenyl-1H-tetrazol-5-yl)thio)methyl)spiro[cyclopropane- 1,5′-inden]-7′(6′H)-one46 152 (R)-6′-hydroxy-2′,4′,6′-trimethyl-3′-(((5-nitro-1H-benzo[d]imidazol-2-yl)thio)methyl)spiro[cyclopropane-1,5′-inden]- 7′(6′H)-one 47 153(R)-3′-(((1H-1,2,4-triazol-3- yl)thio)methyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one 48 154(R)-6′-hydroxy-3′-(((4-hydroxypteridin- 2-yl)thio)methyl)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one 49 155(R)-6′-hydroxy-3′-(((1-(4-hydroxyphenyl)-1H-tetrazol-5-yl)thio)methyl)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one 50 156(R)-4-(5-(((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)thio)-1H-tetrazol-1- yl)phenyl acetate 51 1577′-methyl-4′H-dispiro[cyclobutane-1,6′-indene-5′,2″[1,3]dioxolan]-4′-one 52 1585-hydroxy-2,2,6,8a-tetramethyl- 2,3,3a,8,8a,8b-hexahydro-1H-cyclobuta[d]cyclopenta[b]oxepin-7(5H)-one 53 159((6′R)-3′,6′-dihydroxy-2′,4′,6′-trimethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]- 2′-yl)methyl acetate 54 1605-(((6′R)-2′-(acetoxymethyl)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′- inden]-3′-yl)oxy)-5-oxopentanoic acid55 161 ((6′R)-6′-hydroxy-2′,4′,6′-trimethyl-3′,7′-dioxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane- 1,5′-inden]-2′-yl)methylacetate 56 162 5-(((6′R)-6′-hydroxy-2′,4′,6′-trimethyl-3′,7′-dioxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]-2′-yl)methoxy)-5-oxopentanoic acid 57 163(6′R)-2′-(acetoxymethyl)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]- 3′-yl 2-chloroacetate 58 164(R)-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)methyl2-chloroacetate 59 165 (R)-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)methyl2-morpholinoacetate 60 166 (6′R)-2′-(acetoxymethyl)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′- inden]-3′-yl methyl glutarate 61 167(6′R)-6′-hydroxy-2′-(hydroxymethyl)-2′,4′,6′-trimethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]-3′-yl methyl glutarate 62 168((6′R)-6′-hydroxy-2′,4′,6′-trimethyl-3′,7′-dioxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′- inden]-2′-yl)methyl methyl glutarate63 169 ((6′R)-6′-hydroxy-2′,4′,6′-trimethyl-3′,7′-dioxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]- 2′-yl)methyl 2-chloroacetate64 171 6-(2-hydroxyethyl)-2,5,7-trimethyl-1- methylene-1H-inden-4-ol 65172 6-ethyl-2,5,7-trimethyl-1-methylene-1H-inden-4-ol 66 1732-(4-hydroxy-2,5,7-trimethyl-1-methylene- 1H-inden-6-yl)ethyl acetate 67174 5-(2-hydroxyethyl)-3-(hydroxymethyl)-2,4,6-trimethyl-1H-indene-1,7-diol 68 175(2S,3S,4R,5S,6R)-2-(acetoxymethyl)-6-(((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methoxy)tetrahydro-2H-pyran- 3,4,5-triyl triacetate 69 177(R)-5-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propoxy)-5-oxopentanoic acid 70 178(R)-4-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propoxy)-4-oxobutanoic acid 71 179(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl glycinate 72180 (1a′R,3′R,7′S,7a′R)-3′,7′-dihydroxy-1a′,3′,6′,6′-tetramethyl-6′,7′-dihydro-1a′H-spiro[cyclopropane-1,2′-indeno[3a,4-b]oxiren]-4′(3′H)-one 73 181((1a′R,3′R,6′S,7′S,7a′R)-3′,7′-dihydroxy-1a′,3′,6′-trimethyl-4′-oxo-3′,4′,6′,7′-tetrahydro-1a′H-spiro[cyclopropane-1,2′-indeno[3a,4-b]oxiren]-6′-yl)methyl acetate 74 182(2′R,7′S,7a′S)-2′-chloro-7′-hydroxy- 2′,4′-dimethyl-1′,2′,7′,7a′-tetrahydrospiro[cyclopropane-1,5′-indene]-3′,6′-dione 75 183(2′S,7′S,7a′S)-7′-hydroxy-2′- isopropoxy-2′,4′-dimethyl-1′,2′,7′,7a′-tetrahydrospiro[cyclopropane-1,5′-indene]-3′,6′-dione 76 188(6′S,6″S)-3′,3″-methylenebis(6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one) 77 189(R)-1-((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)-1H-pyrrole-2,5-dione 78 190(R)-1-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)-1H-pyrrole-2,5-dione 79 1916′-hydroxy-4′,6′-dimethylspiro[cyclobutane- 1,5′-inden]-7′(6′H)-one 80192 (R)-2-((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)isoindoline-1,3-dione 81 193(R)-3′-(azidomethyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]- 7′(6′H)-one 82 194(R)-3′-(((R)-3′-(azidomethyl)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′- dihydrospiro[cyclopropane-1,5′-inden]-1′-yl)methyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one 83 195(R)-3′-(3-azidopropyl)-6′-hydroxy-4′,6′-dimethylspiro[cyclopropane-1,5′-inden]- 7′(6′H)-one 84 1963-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl L-prolinate 85197 (R)-2-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)isoindoline-1,3-dione 86 198(R)-6′-hydroxy-2′,4′,6′-trimethyl-3′-((4-nitrophenoxy)methyl)spiro[cyclopropane- 1,5′-inden]-7′(6′H)-one 87 199(R)-6′-hydroxy-2′,4′,6′-trimethyl-3′-(phenoxymethyl)spiro[cyclopropane-1,5′- inden]-7′(6′H)-one 88 200(R)-6′-hydroxy-3′-(2-hydroxybenzyl)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 89 201(R)-N-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane- 1,5′-inden]-3′-yl)propyl)acetamide90 202 (S)-N-(3-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)pyrrolidine-2-carboxamide 91 2033-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propylL-seryl-L-prolinate 92 204 2′-(((tert-butyldimethylsilyl)oxy)methyl)-3′,6′-dihydroxy-2′,4′,6′-trimethyl-2′,3′-dihydrospiro[cyclopropane-1,5′-inden]-7′(6′H)-one 93 205(2′R,3′S,6′R)-3′-amino-2′-(((tert-butyldimethylsilyl)oxy)methyl)-6′-hydroxy-2′,4′,6′-trimethyl-2′,3′-dihydrospiro[cyclopropane-1,5′-inden]-7′(6′H)-one 94 206 (2′R,3′S,6′R)-3′-amino-6′-hydroxy-2′-(hydroxymethyl)-2′,4′,6′-trimethyl-2′,3′-dihydrospiro[cyclopropane-1,5′-inden]-7′(6′H)-one 95 207(S)-2-amino-N-(3-((R)-6′-hydroxy- 2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]- 3′-yl)propyl)-4-methylpentanamide96 208 3-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl(tert-butoxycarbonyl)- L-seryl-L-prolinate 97 2093-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propylL-seryl-L-seryl-L-prolinate 98 2103-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl((S)-2-((S)-2-((S)-2- ((2S,4R)-1-acetyl-4-hydroxypyrrolidine-2-carboxamido)-3-hydroxypropanamido)-3-hydroxypropanamido)-2-cyclohexylacetyl)-L-glutaminyl-L-seryl-L-seryl-L-prolinate 99 212(3′S,6′R)-6′-hydroxy-2′,2′,4′,6′-tetramethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane- 1,5′-inden]-3′-yl L-prolinate100 213 (2′S,3′R,6′R)-2′-(((tert-butyldimethylsilyl)oxy)methyl)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]-3′-yl L- prolinate 101 214(2′S,3′R,6′R)-2′-(((tert-butyldimethylsilyl)oxy)methyl)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]-3′-yl L-seryl-L-seryl-L-prolinate 102 215(2′S,3′R,6′R)-2′-(((tert-butyldimethylsilyl)oxy)methyl)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]-3′-yl ((S)-2-((S)-2-((S)-2-((2S,4R)-1-acetyl-4- hydroxypyrrolidine-2-carboxamido)-3-hydroxypropanamido)-3-hydroxypropanamido)-2-cyclohexylacetyl)-L-glutaminyl- L-seryl-L-seryl-L-prolinate 103 216(3′S,6′R)-6′-hydroxy-2′,2′,4′,6′-tetramethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′- inden]-3′-yl((S)-2-((S)-2-((S)-2-((2S,4R)-1-acetyl-4-hydroxypyrrolidine-2-carboxamido)-3-hydroxypropanamido)-3-hydroxypropanamido)-2-cyclohexylacetyl)-L-glutaminyl-L-seryl-L- seryl-L-prolinate 104 217(R)-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)methyl6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoate 105 218(3′S,6′R)-6′-hydroxy-2′,2′,4′,6′-tetramethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′- inden]-3′-yl6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanoate 106 219(2′S,3′R,6′R)-2′-(((tert-butyldimethylsilyl)oxy)methyl)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]-3′-yl 4-(fluorosulfonyl)benzoate 107 221(S)-2-((3S,6S,9S,12S,15S)-3-((1H-imidazol-4-yl)methyl)-12-(4-aminobutyl)-6,9-bis(hydroxymethyl)-15-isobutyl-1-morpholino-1,4,7,10,13-pentaoxo-2,5,8,11,14- pentaazahexadecan-16-amido)-N1-((S)-1-((3-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)amino)-4-methyl-1- oxopentan-2-yl)pentanediamide 108222 (3′S,6′R)-6′-hydroxy-2′,2′,4′,6′-tetramethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′- inden]-3′-yl4-(fluorosulfonyl)benzoate 109 2233-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl((4R,7S,13S)-13-(2-amino-2-oxoethyl)-7-(3-guanidinopropyl)-6,9,12,15-tetraoxo-1,2-dithia-5,8,14-triazacycloheptadecane-4-carbonyl)glycinate 110 224(R,E)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one oxime 111 225(2′R,3′R,6′R,E)-2′,3′,6′-trihydroxy-2′,4′,6′-trimethyl-2′,3′-dihydrospiro[cyclopropane-1,5′-inden]-7′(6′H)-one oxime 112 226(R)-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)methyl(E)-octadec-9-enoate 113 227 (R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl(E)-octadec-9-enoate 114 228 (2′S,6′R)-6′-hydroxy-2′,4′,6′-trimethyl-2′-((((E)-octadec-9-enoyl)oxy)methyl)-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane- 1,5′-inden]-3′-yl(E)-octadec-9-enoate 115 229 (R,E)-N-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)octadec-9-enamide 116 230N-((3′R,6′R)-6′-hydroxy-2′,2′,4′,6′-tetramethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]-3′-yl)methanesulfonamide 117 231N-((3′R,6′R)-6′-hydroxy-2′,2′,4′,6′-tetramethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]-3′-yl)-4-methylbenzenesulfonamide 118 236(R)-6′-hydroxy-3′-(hydroxymethyl)-1′,2′,4′,6′-tetramethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 119 240(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propylacetoxy(acetyl)carbamate 120 249(R)-6′-hydroxy-2′-(hydroxymethyl)-3′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 121 250((1a′R,2′S,3′R,6′R,7a′S)-3′-acetoxy-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-1′,1a′,2′,3′,6′,7′-hexahydrospiro[cyclopropane-1,5′-cyclopropa[c]inden]-2′- yl)methyl acetate 122 251((1a′S,2′S,3′R,6′R,7a′R)-3′-acetoxy-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-1′,1a′,2′,3′,6′,7′-hexahydrospiro[cyclopropane-1,5′-cyclopropa[c]inden]-2′- yl)methyl acetate 123 252(1a′R,3′S,6′R,7a′S)-3′,6′-dihydroxy-2′,2′,4′,6′-tetramethyl-1′,1a′,2′,3′-tetrahydrospiro[cyclopropane-1,5′-cyclopropa[c]inden]-7′(6′H)-one 124 253(1a′S,3′S,6′R,7a′R)-3′,6′-dihydroxy-2′,2′,4′,6′-tetramethyl-1′,1a′,2′,3′-tetrahydrospiro[cyclopropane-1,5′-cyclopropa[c]inden]-7′(6′H)-one 125 254(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl4-sulfamoylbenzoate 126 255 (R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl sulfamate 127256 3-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl1-(3-((R)-6′-hydroxy- 2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)-1H-1,2,3-triazole-4- carboxylate 128 2573-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl1-((R)-6′-hydroxy- 2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)-1H-1,2,3-triazole-4- carboxylate 129 258(4-carboxy-4-(4-carboxy-4-((3-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)amino)butanamido)butanoyl)glutamic acid 130 259(R)-3′-((S)-2,2-dioxido-1,2,3- oxathiazinan-4-yl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one 131 262 methyl(R)-2-(((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)sulfonyl)acetate 132 263 methyl2-((((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)sulfinyl)acetate 133 267(R)-2-amino-N-((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)-N-methoxyacetamide 134 268(R)-2,2,2-trifluoro-N-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)acetamide 135 269(R)-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)methyl(4-methoxyphenyl)sulfamate 136 270(R)-3′-(aminomethyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]- 7′(6′H)-one 137 272(5S,6S,7S)-3-(((3-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propanoyl)oxy)methyl)-8-oxo-7-(2-(thiophen-2-yl)acetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5-oxide 138 273(5S,6S,7S)-3-((((3-((R)-6′-hydroxy- 2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)carbamoyl)oxy)methyl)-8-oxo-7-(2-(thiophen-2-yl)acetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2- carboxylic acid 5-oxide 139 274(6S,7S)-3-((((3-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)carbamoyl)oxy)methyl)-8-oxo-7-(2-(thiophen-2-yl)acetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2- carboxylic acid 5,5-dioxide 140275 N-(((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)pyrrolidine-2-carboxamide 141 2762-amino-N-(((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)-4-methylpentanamide 142 284(R)-3-((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)pyrimidine-2,4(1H,3H)-dione 143 285N-hydroxy-N-[(6′-hydroxy-2′,6′-dimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl]sulfuric diamide 144 286(R)-5-fluoro-1-((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)pyrimidine-2,4(1H,3H)-dione 145 287(R)-5-fluoro-3-((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)pyrimidine-2,4(1H,3H)-dione 146 289(R)-1-((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)pyrimidine-2,4(1H,3H)-dione 147 290((S)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)methyl5-oxo-5-(((S)-1,2,3,10- tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)amino)pentanoate 148 2913-((S)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl5-oxo-5-(((S)-1,2,3, 10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)amino)pentanoate 149 292N1-(((S)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)-N5-((S)-1,2,3,10- tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)glutaramide 150 2933-((S)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)-N-((S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)propanamide 151 2942-amino-N-(((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)-N-methoxypropanamide 152 2952-amino-N-(((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)-N-methoxy-4- methylpentanamide 153 2962-amino-N-(((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)methyl)-N-methoxy-4-(methylthio)butanamide 154 2972-amino-N-(((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)-3-(1H-indol-3-yl)-N- methoxypropanamide 155 298(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl(tert-butoxycarbonyl)glycinate 156 299(2′S,3′R,6′R)-2′-(azidomethyl)-3′,6′-dihydroxy-2′,4′,6′-trimethyl-2′,3′-dihydrospiro[cyclopropane-1,5′-inden]-7′(6′H)-one 157 300(2′R,3′R,6′R)-3′-azido-6′-hydroxy-2′-(hydroxymethyl)-2′,4′,6′-trimethyl-2′,3′-dihydrospiro[cyclopropane-1,5′-inden]-7′(6′H)-one 158 301(R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl6-oxo-6-phenylhexanoate 159 302 ((2′S,3′R,6′R)-3′,6′-dihydroxy-2′,4′,6′-trimethyl-7′-oxo-2′,3′,6′,7′- tetrahydrospiro[cyclopropane-1,5′-inden]-2′-yl)methyl 3,5-dinitrobenzoate 160 303(2′S,3′R,6′R)-2′-(((3,5-dinitrocyclohexa-2,4-diene-1-carbonyl)oxy)methyl)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′- inden]-3′-yl 3,5-dinitrobenzoate 161304 (R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl propiolate162 305 (R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl(4-nitrophenyl) carbonate 163 306(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl4-methylbenzenesulfonate 164 307(3′R,6′R)-3′-azido-6′-hydroxy-2′,2′,4′,6′-tetramethyl-2′,3′-dihydrospiro[cyclopropane-1,5′-inden]-7′(6′H)-one 165 308(3′R,6′R)-3′-amino-6′-hydroxy-2′,2′,4′,6′-tetramethyl-2′,3′-dihydrospiro[cyclopropane-1,5′-inden]-7′(6′H)-one 166 309(2′R,3′S,6′R)-3′-azido-2′-(((tert-butyldimethylsilypoxy)methyl)-6′-hydroxy-2′,4′,6′-trimethyl-2′,3′-dihydrospiro[cyclopropane- 1,5′-inden]-7′(6′H)-one 167310 (R)-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)methyl4-sulfamoylbenzoate 168 311(3′S,6′R)-6′-hydroxy-2′,2′,4′,6′-tetramethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′- inden]-3′-yl4-sulfamoylbenzoate 169 312 (R)-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)methyl4-methylbenzenesulfonate 170 313 2,3,4,5,6-pentafluoro-N-((3′R,6′R)-6′-hydroxy-2′,2′,4′,6′-tetramethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]-3′-yl)benzenesulfonamide 171 314(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl4-methylbenzenesulfonate 172 315 (R)-3′-((2,5-dimethyl-1H-pyrrol-3-yl)methyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one 173 316((2′5,3′R,6′R)-3′,6′-dihydroxy- 2′,4′,6′-trimethyl-7′-oxo-2′,3′,6′,7′-tetrahydrospiro[cyclopropane-1,5′-inden]- 2′-yl)methyl4-(fluorosulfonyl)benzoate

TABLE XII shows previously identified Illudin analogs. # Analog IUPACName of Illudofulvene Analog 1 001(R)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one 2 002 (6′R)-6′-hydroxy-3′-(hydroxymethyl)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 3 003(6′R,6′″R)-3′,3′″-methylenebis(6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one) 4 004(R)-3′-bromo-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)- one 5 005(R)-6′-hydroxy-3′-iodo-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one 6 006(R)-6′-hydroxy-3′-(4-hydroxybenzyl)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 7 007(R)-6′-hydroxy-3′-(4-methoxybenzyl)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 8 008(R)-((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)methoxy)methylacetate 9 009 (R)-6′-hydroxy-3′-(3-hydroxypropyl)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 10 010(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propanal 11 011(R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- indene]-3′-carbaldehyde 12 012(R)-6′-hydroxy-2′,4′,6′-trimethyl-3′-nitrospiro[cyclopropane-1,5′-inden]-7′(6′H)-one 13 0134-hydroxy-5-(((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)cyclohexane-1,3-dicarbaldehyde 14 014(4a′S,7′R,9b′S)-7′-hydroxy-4a′,7′,9′-trimethyl-4a′,9b′-dihydro-4′H-spiro[cyclopropane-1,8′-indeno[1,2-d][1,3]dioxin]-6′(7′H)-one 15 015(R)-3′-(hydroxymethyl)-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane- 1,5′-inden]-6′-yl acetate 16 016(R)-3′-(ethoxymethyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]- 7′(6′H)-one 17 017(6′R,6R)-3′,3″′-(oxybis(methylene))bis(6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane- 1,5′-inden]-7′(6′H)-one)18 018 (R)-6′-hydroxy-2′,4′,6′-trimethyl-3′-((((2R,3S,4R,5R,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)spiro[cyclopropane-1,5′- inden]-7′(6′H)-one 19 019(6′R)-3′4(2,3-dihydroxypropoxy)methyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane- 1,5′-inden]-7′(6′H)-one 20020 (R)-3′-((2-bromoethoxy)methyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane- 1,5′-inden]-7′(6′H)-one 21 021(R)-6′-hydroxy-3′-(((2-methoxypropan- 2-yl)oxy)methyl)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one 22 022(R)-6′-hydroxy-3′-((2-hydroxyethoxy)methyl)-2′,4′,6′-trimethylspiro[cyclopropane- 1,5′-inden]-7′(6′H)-one 23 023(R)-6′-hydroxy-3′-(((4-hydroxyphenyethio)methyl)-2′,4′,6′-trimethylspiro[cyclopropane- 1,5′-inden]-7′(6′H)-one 24 024(R)-3′-((benzylthio)methyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 25 025methyl (R)-2-(((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]- 3′-yl)methyl)thio)acetate26 026 (R)-6′-hydroxy-2′,4′,6′-trimethyl-3′-((p-tolylthio)methyl)spiro[cyclopropane-1,5′- inden]-7′(6′H)-one 27 027(R)-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)methyl phenylcarbonate 28 028 (R)-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)methyl benzoate29 029 (R)-2-(((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)thio)acetic acid 30 030 methyl(R)-2-(((6′-hydroxy-1′-((2-methoxy-2-oxoethyl)thio)-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)thio)acetate 31 031 methyl2-((((6′R)-6′,7a′-dihydroxy-1′- ((2-methoxy-2-oxoethyl)thio)-2′,4′,6′-trimethyl-7′-oxo-1′,6,7′,7a′-tetrahydrospiro[cyclopropane-1,5′-inden]-3′- yl)methypthio)acetate 32032 (6′R)-3′-(((2,3-dihydroxypropyl)thio)methyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane- 1,5′-inden]-7′(6′H)-one33 033 7′-methyl-4′H-dispiro[cyclopropane-1,6′-indene-5′,2″-[1,3]dioxolan]-4′-one 34 034(R)-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)methyl acetate 35 0356′-hydroxy-4′-methylspiro[cyclopropane- 1,5′-inden]-7′(6′H)-one 36 036(R)-3′-((1H-imidazol-1-yl)methyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane- 1,5′-inden]-7′(6′H)-one 37037 1-carboxy-2-((((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)thio)ethan-1-aminium 38 038(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propanoic acid 39 039(R)-3′-(3,3-dimethoxypropyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 40 040(R)-3′-(3,3-diethoxypropyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 41 041(R,Z)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)acrylaldehyde 42042 (R)-3′-(hydroxymethyl)-4′,6′-dimethyl-6′-((triethylsilypoxy)spiro[cyclopropane-1,5′- inden]-7′(6′H)-one 43 043(R)-6′-hydroxy-2′,4′,6′-trimethyl-3′-(((triethylsilyl)oxy)methyl)spiro[cyclopropane- 1,5′-inden]-7′(6′H)-one44 044 (R)-2′,4′,6′-trimethyl-6′-((triethylsilyl)oxy)-3′-(((triethylsilyl)oxy)methyl)spiro[cyclopropane- 1,5′-inden]-7′(6′H)-one45 045 methyl 2-((7-hydroxy-5-(2-hydroxyethyl)-3-(hydroxymethyl)-2,4,6-trimethyl-1H- inden-1-yl)thio)acetate 46 046(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl acetate 47047 (6′R)-3′-(2-(1,7-dihydroxy-2,4,6-trimethyl-1H-inden-5-yl)ethyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one 48 048(R)-6′-hydroxy-2′,4′,6′-trimethyl-1′-(p-tolylthio)-3′-((p-tolylthio)methyl)spiro[cyclopropane- 1,5′-inden]-7′(6′H)-one 49049 (R)-6′-hydroxy-2′,4′,6′-trimethyl-3′-(p-tolylthio)spiro[cyclopropane-1,5′-inden]- 7′(6′H)-one 50 050(R)-6′-hydroxy-2′,4′,6′-trimethyl-1′,3′-bis(p-tolylthio)spiro[cyclopropane-1,5′- inden]-7′(6′H)-one 51 051(R)-2-(2-(((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)thio)acetoxy)ethyl 2-mercaptoacetate 52 052ethane-1,2-diyl bis(2-((((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]- 3′-yl)methyl)thio)acetate) 53 053(R)-3′-((2-(2-bromoethoxy)ethoxy)methyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane- 1,5′-inden]-7′(6′H)-one 54 054(R)-6′-hydroxy-1′-(((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)-3′-(hydroxymethyl)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one 55 0555-(2-hydroxyethyl)-1-((4-hydroxyphenyl)thio)-3-(((4-hydroxyphenyl)thio)methyl)- 2,4,6-trimethyl-1H-inden-7-ol 56 056(R)-6′-hydroxy-3′-((4-hydroxyphenyl)thio)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 57 057(R)-6′-hydroxy-1′-((4-hydroxyphenyl)thio)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 58 058(R)-6′-hydroxy-1′,3′-bis((4-hydroxyphenyl)thio)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one 59 059(6′S,7′R)-4′-methyl-6′-((triethylsilypoxy)-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-7′-ol 60 060(R)-7′-methyl-4′H-dispiro[cyclopropane-1,6′-indene-5′,2″-[1,3]dioxolan]-4′-ol 61 061(S)-4′-methyl-6′-((triethylsilyl)oxy)spiro[cyclopropane-1,5′-inden]-7′(6′H)-one 62 062 (R)-6′-hydroxy-2′-(hydroxymethyl)-4′,6′-dimethylspiro[cyclopropane-1,5′-inden]- 7′(6′H)-one 63 063(R)-6′-hydroxy-2′,3′-bis(hydroxymethyl)-4′,6′-dimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 64 064N-acetyl-S-(((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)-L-cysteine 65 065(R)-2-acetamido-3-((((R)-6′-hydroxy-4′,6′-dimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)thio)-N-((S)-1- phenylethyl)propanamide 66 066(S)-2-acetamido-3-((((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)thio)-N-((S)-1- phenylethyl)propanamide 67 0674-methyl-2,3-dihydro-5H-indeno[5,6-b]furan-5-one 68 0685-hydroxy-6-(2-hydroxyethyl)-7-methyl-1H-inden-1-one 69 0695-(2-hydroxyethoxy)-6-(2-hydroxyethyl)-7-methyl- 1H-inden-1-one 70 070(3a′R,4′R)-4′-hydroxy-7′-methyl-3a′,4′-dihydro-1′H-dispiro[cyclopropane-1,6′-indene-5′,2″-[1,3]dioxolan]-1′-one 71 071(3a′R,4′R)-7′-methyl-4′-((triethylsilyl)oxy)-3a′,4′-dihydro-1′H-dispiro[cyclopropane-1,6′-indene-5′,2″-[1,3]dioxolan]-1′-one 72 072(7′R,7a′R)-7′-hydroxy-4′-methyl-7′,7a′-dihydrospiro[cyclopropane-1,5′-indene]-3′,6′- dione 73 073 (7′R,7a′R)-4′-methyl-7′-((triethylsilyl)oxy)- 7′,7a′-dihydrospiro[cyclopropane-1,5′- indene]-3′,6′-dione 74 074(6′R)-3′-((((2,2-dimethyl-1,3-dioxolan-4-yl)methypthio)methyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one 75 075(R)-(6′-hydroxy-4′,6′-dimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]- 2′-yl)methyl acetate 76 076(R)-(6′-hydroxy-4′,6′-dimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-indene]- 2′,3′-diyl)bis(methylene)diacetate 77 077 (R)-(6′-hydroxy-3′-(hydroxymethyl)-4′,6′-dimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-2′-yl)methyl acetate 78 078(R)-(6′-hydroxy-2′-(hydroxymethyl)-4′,6′-dimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane- 1,5′-inden]-3-yl)methyl acetate79 079 (R)-6′-hydroxy-2′-(methoxymethyl)-4′,6′-dimethylspiro[cyclopropane-1,5′-inden]- 7′(6′H)-one 80 080(R)-6′-hydroxy-3′-(methoxymethyl)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]- 7′(6′H)-one 81 081(R)-6′-hydroxy-2′-(hydroxymethyl)-3′-(methoxymethyl)-4,6′-dimethylspiro[cyclopropane- 1,5′-inden]-7′(6′H)-one82 082 (R)-6′-hydroxy-2′,3′-bis(methoxymethyl)-4′,6′-dimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 83 083(R)-2-acetamido-3-((((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3-yl)methyl)thio)-N-((S)-1-((2-(((S)-4-methyl-1-oxopentan-2-yl)amino)- 2-oxoethyl)amino)-1-oxo-3-phenylpropan-2- yl)propanamide 84 084(S)-2-((R)-2-acetamido-3-((((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3-yl)methyl)thio)propanamido)-4-methyl-N-(2-oxo-2-(((R)-1-oxo-3-phenylpropan-2- yl)amino)ethyl)pentanamide 85 085(S)-2-((R)-2-acetamido-3-((((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′- dihydrospiro[cyclopropane-1,5′-inden]-3-yl)methyl)thio)propanamido)-4-methyl-N-((S)-4-methyl-1-oxo-1-(((R)-1-oxo- 3-phenylpropan-2-yl)amino)pentan-2-yl)pentanamide 86 086 (R)-2-acetamido-3-((((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3-yl)methyl)thio)-N-(2-oxo-2-(((R)-1-oxo-3-phenylpropan-2-yl)amino)ethyl)propanamide 87 087(S)-2-((R)-2-acetamido-3-((((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3-yl)methyl)thio)propanamido)-4-methyl-N-((R)-4-methyl-1-(((S)-4-methyl-1- oxopentan-2-yl)amino)-1-oxopentan-2-yl)pentanamide 88 088 (R)-(6′-acetoxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)methyl acetate 89089 N5-((R)-1-((carboxymethyl)amino)-3-((((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3-yl)methyl)thio)-1-oxopropan-2-yl)- D-glutamine 90 090(R)-2′-(hydroxymethyl)-4′,6-dimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-6′-yl acetate 9 094(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl acetate 95095 (R)-6′-hydroxy-3′-(3-methoxypropyl)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- inden]-7′(6′H)-one 96 096(R)-6′-hydroxy-2′,4′,6′-trimethyl-3′- (((1-methyl-1H-imidazol-2-yl)thio)methyl)spiro[cyclopropane- 1,5′-inden]-7′(6′H)-one 97 097S-(((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5-inden]-3′-yl)methyl)homocysteine 98 098((S)-3-((((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)thio)-2- methylpropanoyl)proline 99 099(2′S,6′R)-6′-hydroxy-2′-(hydroxymethyl)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′- indene]-3′,7′(2′H,6′H)-dione100 100 S-(((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)-N-S-(((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)cysteinyl-L-asparaginylglycyl- L-arginylcysteine 101 101S-(((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)-N-S-(((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)cysteinyl-L-arginylglycyl-L- asparaginylcysteine 102 102S-(((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)cysteinyl-L-asparaginylglycyl- L-arginylcysteine 103103 (R)-(6′-acetoxy-2′-(hydroxymethyl)-4′,6′-dimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane- 1,5′-inden]-3′-yl)methyl acetate104 104 (R)-8′-hydroxy-6′,8′-dimethyl-1′,5′-dihydrospiro[cyclopropane-1,7′-indeno[1,2- e][1,3]dioxepin]-9′(8′H)-one105 105 (E)-2-((2R,4S)-4-hydroxy-2-((1R,2S)-2-hydroxy-4,4-dimethylcyclopentyl)-2- methylcyclobutylidene)propanal 106132 (E)-7-(chloromethylene)-5-hydroxy-5,9-dimethylspiro[3.5]non-8-en-6-one 107 1705-(2-hydroxyethyl)-3-(hydroxymethyl)- 2,4,6-trimethyl-1H-inden-7-ol 108176 3-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl leucinate 109184 (R)-1-hydroxy-1-((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)urea 110 185(S)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]-7′(6′H)-one 111 186(6′S,7′R)-6′,7′-dihydroxy-2′,4′,6′-trimethyl-7′,7a′-dihydrospiro[cyclopropane-1,5′- inden]-3′(6′H)-one 112 187(S)-6′-hydroxy-3′-(hydroxymethyl)-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]- 7′(6′H)-one 113 211(R)-3′-(3-aminopropyl)-6′-hydroxy-2′,4′,6′-trimethylspiro[cyclopropane-1,5′-inden]- 7′(6′H)-one 114 220(R)-1-acetoxy-1-((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane- 1,5′-inden]-3′-yl)methyl)ur 115232 (R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propylhydroxycarbamate 116 233 ethyl(R)-hydroxy((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)carbamate 117 234 benzyl(R)-hydroxy((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)carbamate 118 235 tert-butyl(R)-hydroxy((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)carbamate 119 237(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl(2-bromoethyl)carbamate 120 238 (R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl(2-chloroethyl)carbamate 121 239(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl(2-hydroxyethyl)carbamate 122 241(R)-N-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)methanesulfonamide 123 242(R)-N-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)-4-methylbenzenesulfonamide 124 243(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl(2-fluoroethyl)carbamate 125 244(R)-1-hydroxy-1-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)urea 126 245(R)-1-hydroxy-1-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)thiourea 127 246(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propylmorpholine-4-carboxylate 128 247(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propylmorpholine-4-carboxylate 129 248(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl[1,4′-bipiperidinel-1′-carboxylate 130 260(R)-N-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3-yl)propyl)-[1,4′-bipiperidine]-1′-carboxamide 131 261(R)-3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl1H-imidazole-1-carboxylate 132 264N-[3-(6′-hydroxy-2′,6′-dimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]- 3′-yl)propyl]sulfuricdiamide 133 265 N-hydroxy-N′-[3-(6′-hydroxy-2′,6′-dimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′- inden]-3′-yl)propyl]sulfuric diamide 134266 N-[3-(6′-hydroxy-2′,6′-dimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl]-N-methoxysulfuric diamide 135 271(R)-N-((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)methanesulfonamide 136 277(R)-1-hydroxy-3-((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane- 1,5′-inden]-3′-yl)methyl)urea137 278 (R)-1-((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)-3-methoxyurea 138 279(R)-1-((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)-3-(2-hydroxyethyl)urea 139 280(R)-1-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)-3-(2-hydroxyethyl)urea 140 281(R)-1-(2-chloroethyl)-3-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)urea 141 282(R)-1-(2-chloroethyl)-3-((6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)urea 142 283N-[(6′-hydroxy-2′,6′-dimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′- yl)methyl]sulfuricdiamide 143 288 (R)-1-hydroxy-3-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propyl)urea

TABLE XIII Summary NCI DTP 60 Cell Line Data. Mean GI50 Mean TGI MeanLD50 NAME/NSC inhibition cytostatic cytotoxic Pyrrolobenzodiazepines 7nM 302 nM >23,000 nM* 694501 Maytansine** 19 nM 318 nM 49,200 nM 153858Fumagillol 6,130 nM 9,850 nM >50,000 nM 642492 Dolstatin-10 17 nM 2,680nM >50,000 nM 376128 Auristatins 1.4 nM 902** nM >5,000 nM** 654663Enadiyne 157365 2,900 nM >100,000 nM >100,000 nM Halichondrin B 1.2 nM199 nM >1,000 nM 609395 Tubulysin A 12 nM 1,318 nM >10,000 nM Illudin S10 nM 64 nM 511 nM Illudin M 3 nM 20 nM 912 nM

TABLE XIV Mechanisms of Drug Resistance. Mechanism of Resistance toSyn-Illudins, Multi-drug Resistance Syn-illudins, and AcylfulvenesGp170/MDR1 No Gp180/MRP No Topoisomerase I No Topoisomerase II NoMVP/LRP (vault) No Thiol content/GST pi No DNA repair No Myc expressionNo Bcl-2 expression No BRCA status No P53 status No P21 status No MGMTexpression No Microtubulin alteration No

TABLE XV ability of Illudin, Syn-Illudin & Acylfulvene to inhibit tumorcell growth. Mean IC50 value (nM) ± SD, N = 3 unless otherwise indicatedAnalog MV522 Target Cell Line 8392B Nontarget Cell Line Number 2 hrexposure 48 hr exposure 2 hr exposure 48 hr exposure 001 2200 ± 100 350± 20  830 ± 100 002 110 ± 40  70 ± 10 26000 ± 4500  800 ± 100 004  4200   600 008 870 ± 90 630 ± 80 12200 ± 700  15100 ± 2200 009 500 ± 30  850± 180  47100 ± 11000 43200 ± 2300 010  8900 ± 1500 170 ± 60 29400 ± 160014500 ± 1700 011 4900 ± 900 1200 (N = 2) >100000  40400 ± 6700 012  5150± 1350 320 ± 90 42200 ± 5000 18800 ± 2800 013 5100 ± 700  270 ± 13011900 ± 1300 4200 ± 400 014 115 ± 30  460 ± 120 9650 ± 200 1100 ± 300015 1800 ± 200  480 ± 110  810 ± 260 1300 ± 150 016  490 ± 130 440 ±90 >100000  870 ± 60 017 2400 ± 360 320 ± 60 14700 ± 900  018  8800 ±2900  4200 ± 1300 019 470 ± 60 660 ± 80 >75000 020  530 ± 140 230 ± 1025000 ± 3100 021  2400 ± 1000  930 ± 250 34400 ± 9400 022  700 ± 200 680 ± 180 31700 ± 1400 023  2900 ± 1140 2750 ± 500 >138000  024 1800 ±200 1200 ± 300 12800 ± 2100 025 1300 ± 310 1200 ± 100 >25000 030   >3000031   >3000 032  600 ± 190 210 ± 30 >30000 033 10000 ± 1100 4600 ± 20029900 ± 3300 034 1400 ± 170 490 ± 40 >100000  4400 ± 200 035 5600 ±600 >150000  037 26000 ± 5000 29200 ± 2300 >85000 038 750 ± 60 24900 ±8000 039 1500 ± 240 600 ± 40 24600 ± 2400  820 ± 250 040 3400 ± 360 700± 90 24000 ± 3300 5200 ± 470 060 19400 ± 1800 27600 ± 3000 062 2600 ±300  660 ± 200 37100 ± 2300 063 43000 ± 5700  580 ± 250 064 28000 ± 46001200 ± 300 065  6200 ± 1100  2500 ± 1200 075 19600 ± 9700 62000 ± 3600076 24000 ± 6100 39500 ± 7200 077  9200 ± 1200 078 20400 ± 6300 >100000 079  7700 ± 3500 >100000  080  8800 ± 2400 >100000  081 >80000 >80000082 50600 ± 7100 >100000  083 37200 ± 2900 >42000  084 28200 ±1400 >42000  085 >40000   >40000 086 087 >40000 24700 ± 3900 >40000 088089 19300 ± 5700 15500 ± 2800 >60000 090 2500 ± 400 2900 ± 400 1600 ±200 3800 ± 300 094  800 ± 100 210 ± 20  9000 ± 1700 110 ± 10 096 2700 ±400 6200 ± 600 >88000  >3000 097 2900 ± 100 >82000 098 18800 ± 2500 4600± 250 >65000 11700 ± 1800 099  8400 ± 1100 1800 ± 200 4000 ± 400 300 ±20 100 >10000 1700 ± 500 101  >8000   >7500 102 >13000 1300 ± 100 10331800 ± 4900 5900 ± 400 12100 ± 2000 2300 ± 200 104 6300 ± 400 6000 ±500 36400 ± 6500 2700 ± 600 105  7300 ± 1200 2100 ± 400 >100000  106 5200 ± 1000 >83000 107 >50000 1600 ± 100 >50000 108 12300 ± 2300 520 ±50 >55000  6000 ± 1600 109 >50000 >50000 110 >55000 1400 ± 100 >5500025300 ± 2100 111 16700 ± 2100 11900 ± 2800 34600 ± 2100 10200 ± 1000 11210000 ± 2000  6700 ± 1200 14900 ± 100  5200 ± 300 113 85000 ± 700  14100± 3000 >93000  7800 ± 1000 114 1500 ± 100 260 ± 70 25100 ± 1000  700 ±100 115 1500 ± 100 70 ± 5 1600 ± 700 630 ± 60 116  400 ± 100 1000 ± 50 7000 ± 400 170 ± 30 117 1100 ± 100 100 ± 30  7900 ± 1600 10 ± 2 11814000 ± 2000  740 ± 120 24500 ± 4500 2000 ± 400 119 1100 ± 70  270 ±40 >33000 >10000  120 2800 ± 900  600 ± 100 19100 ± 4600  510 ± 110 121300 ± 10  90 ± 10 15200 ± 6000 1300 ± 500 122 6400 ± 300 2400 ± 30014500 ± 1200 1100 ± 300 123 1900 ± 400 600 ± 60 450 ± 30 2400 ± 500 1242800 ± 700  870 ± 350 >30000 2400 ± 550 125 3700 ± 600 1200 ± 200 15500± 1400  600 ± 100 126 2100 ± 500  900 ± 100 >30000 330 ± 80 127 870 ± 30340 ± 90 >30000 100 ± 40 128  840 ± 230 370 ± 50 >35000 800 ± 70129 >136000  19700 ± 1900 >136000  39400 ± 9200 130  700 ± 100 130 ± 4027,000 ± 7000  4400 ± 500 133 58800 ± 6600 15800 ± 2600 12200 ± 23002700 ± 400 134 50000 ± 6000 28000 ± 4000 43900 ± 5100  8500 ± 2000 1351600 ± 300 22 ± 4  70 ± 20 22 ± 2 136 430 ± 10 130 ± 10  >6200 25 ± 2137  850 ± 110 1200 ± 100  8500 ± 1200 710 ± 60 138 2100 ± 200 1000 ±200 5400 ± 200  820 ± 230 139 6400 ± 900 3400 ± 500 11600 ± 900   2600 ±1000 140 17100 ± 5100   >14000 12700 ± 300  >14000  141 11400 ± 10003700 ± 800 13700 ± 1900 1100 ± 140 142  90 ± 10 24 ± 7  6400 ± 1100 80 ±6 143  43500 ± 11300 11400 ± 1800  56500 ± 20000 3600 ± 700 146 2500 ±400  740 ± 280 13,000 ± 1200  147 >76000  26100 ± 12900 >76000 43800 ±3000 148 17100 ± 1100  6800 ± 1100  61000 ± 11600  6700 ± 1600 149  2900± 1000 1500 500 44600 ± 1400 4100 ± 900 150  9500 ± 1600 1400 ± 40059000 ± 5500 10600 ± 800  151 7900 ± 400  4200 ± 1600 25500 ± 1200  6600± 2300 152  6400 ± 1200 49000 ± 7700 9100 ± 100 153  8700 ± 2700 10900 ±3400 >90000 15800 ± 9600 154 >70000  61300 ± 10000 >70000  46,700 ±13100 155  8200 ± 1200 3600 ± 400 17,000 ± 4000   9100 ± 1100 156 7200 ±500 3100 ± 100 32,300 ± 9,400  5500 ± 1200157 >400,000   >123,000 >350,000   13100 ± 1600158 >175,000   >175,000 >200,000   61,000 ± 9,000 159 2700 ± 400 120 ±10 13,700 ± 4,200 <10 nM 160 1900 ± 200  500 ± 200  52,400 ± 17,800 3200 ± 1100 161 2800 ± 500 3300 ± 700 13,800 ± 3,400 >10,000  163 3500± 800 820 ± 40 18600 ± 800   910 ± 100 164  70 ± 10  3500 ± 1600 130 ±40 165  7700 ± 1100 290 ± 40 11000 ± 3300 11000 ± 1000 166 6500 ± 600 7200 ± 1900  6500 ± 2100  6000 ± 1500 167 14800 ± 2200 18500 ± 2300 1697100 ± 600 2300 ± 600 177 7500 ± 800 1900 ± 800 73000 ± 5000  4100 ±1300 178 21000 ± 4000 1000 ± 100 32000 ± 9000  >8000 180 19900 ± 300   >4000  5200 ± 1800 660 ± 50 182  99000 ± 12000 38000 ± 8200 39000 ±7000 18700 ± 2700 183 >120,000   >275,000 >120,000   >235,000  184  800± 300 210 ± 20 >100,000   >10000  185 1700 ± 600 1900 ± 100 186 144000 ±32000  70000 ± 16000  79000 ± 24000 48000 ± 2000 187 1300 ± 400  900 ±200 3200 ± 800 3200 ± 700 189  8900 ± 2500  6100 ± 2600 41,000 ± 3700 190 19,000 ± 4000   >9,000 56,000 ± 2000  >9,000 191 >140,000    49,000± 13000 >140,000   15000 ± 4000 192 1,600 ± 200   700 ± 100  8700 ± 1700200 ± 30 193 1400 ± 400 2500 ± 600 48,000 ± 7000  >11,000  195 1400 ±200  390 ± 120 21,000 ± 6000   4300 ± 1200 196  840 ± 100  450 ± 12080,000 ± 5000  >9,200 197 950 ± 70  500 ± 100 9500 ± 400 11,300 ± 100 198  700 ± 100 2800 ± 600  >8,200 >82,000  199 4700 ± 600  2500 ±1100 >93,000  >9,300 201  360 ± 110 260 ± 70 13,000 ± 1700  26,000 ±7000  202 1200 ± 100  650 ± 100 >62,000   >6200 203  760 ± 170  940 ±330 48,000 ± 6000   >5500 204 220 ± 40 1600 ± 300 4100 ± 800 8600 ± 800205  8400 ± 2200 1200 ± 400 >185,000   >2,600 206 610 ± 40 230 ± 2020,000 ± 1000  8200 ± 200 207 570 ± 60 410 ± 60 208 1200 ± 100  930 ±160 25,000 ± 3000  209  3900 ± 1100  610 ± 100 >90,000  210 40,000 ±4000  5500 ± 600 211  470 ± 120  430 ± 100 59,000 ± 9000  212  80 ± 1055 ± 5 213 2300 ± 700 1700 ± 700 214 2900 ± 800 360 ± 30 215 26,000 ±3000   490 ± 120 216 460 ± 60 150 ± 40 217 2,200 ± 100  2,200 ± 100 43,000 ± 4,000 >7,000 218 10,000 ± 3,000  600 ± 200 15,000 ± 6,000  600± 100 219 >52,000  >52,00 >52,000  >52,000  220  90 ± 10 130 ± 10101,000 ± 18,000 40,000 ± 3,000 221 >21,000  2,500 ±200  >21,000  >21,000  222 5,000 ± 100  1,100 ± 100  9,300 ± 200  330 ±60 223 20,000 ± 3,700 2,700 ± 300  >185,000   >55,000 224 >200,000   >130,000 >200,000   >130,000  225 47,000 ± 4,000  55,000± 11,000 >350,000    33,000 ± 13,000 226 >59,000  >59,000 >59,000  >59,000  227 >57,000  4,400 ± 700  >57,000  16,000 ±4,000 228 >38,000   >38,000 24,000 ± 3,000 >38,000  229 >56,000  >2,000 >56,000  >2,000 230 620 ± 80 100 ± 10 38,000 ± 5,000 1,000 ±200  231 1,500 ± 100  280 ± 10 14,000 ± 4,000 232  700 ± 100 460 ± 6042,000 ± 6,000 3,300 ± 600  233 3,200 ± 300  350 ± 80 >150,000   2,400 ±700  234 3,000 ± 300  1,100 ± 400  24,000 ± 6,000  9,000 ± 1,000 2353,500 ± 400  2,200 ± 400  49,000 ± 6,000  6,500 ± 1,600 236  49,000 ±11,000 29,000 ± 5,000  48,000 ± 10,000 237 1,200 ± 300   730 ± 14022,000 ± 1,000 6,600 ± 900  238  780 ± 190 57 ± 8 23,000 ± 2,000  4,700± 1,200 239 420 ± 60  70 ± 20 39,000 ± 3,000 28,000 ± 4,000 240 2,900 ±100  1,300 ± 200  >24,000  1,300 ± 100  241 560 ± 90 110 ± 20 >28,000 18,000 ± 4,000 242 2,400 ± 400   580 ± 150 18,000 ± 2,000 2,900 ± 600 243 2,200 ± 500   670 ± 240  64,000 ± 10,000 26,000 ± 6,000 244 1,600 ±400  150 ± 10  87,000 ± 11,000 35,000 ± 7,000 245  3,400 ± 1000 440 ± 9079,000 ± 7,000 14,000 ± 1,700 246 2,800 ± 260  1,900 ± 450  14,000 ±2,000  6,200 ± 1,300 247  6,100 ± 2,000 1,200 ± 250  10,000 ± 1,400 7,100 ± 1,700 248  830 ± 100 200 ± 25 23,000 ± 1,000  610 ± 120 2494,100 ± 820   420 ± 100 18,000 ± 3,500 19,000 ± 3,800 250  99,000 ±21,000 137,000 ± 14,000 >275,000   137,000 ± 10,000 251 128,000 ± 4,000 51,000 ± 1,000 >275,000   82,000 ± 8,000 252 >380,000   33,000 ±3,000 >380,000   >380,000  253 >380,000    >38,000 >380,000   >380,000 254 2,700 ± 800  1,100 ± 100  43,000 ± 6,000 >65,000  255 2,900 ± 500 55 ± 2 119,000 ± 15,000 99,000 ± 4,000 256 1,500 ± 200   880 ± 200 7,500± 800  7,100 ± 300  257 2,800 ± 600  320 ± 30 25,000 ± 2,000 26,000 ±3,000 258 >45,000   >45,000 >45,000  >45,000  259 16000 ± 3000 2400 ±200 >85,000  4700 ± 400 260 1600 ± 500 150 ± 20 >64,000  19000 ± 4500261  6300 ± 1100 1000 ± 150 64000 ± 2000 38000 ± 2100 262  8700 ± 13003900 ± 570 287000 ± 14000  73000 ± 17000 263 2000 ± 300 1400 ± 200124000 ± 18000 39000 ± 7000 264 1400 ± 100  76 ± 17 >85,000   54000 ±20000 265 810 ± 20  8 ± 1 1100 ± 200 250 ± 80 266 140 ± 20  70 ± 18 56000 ± 15000 32000 ± 7000 267  900 ± 160 160 ± 20 >90,000  28000 ±8000 268 2100 ± 200 330 ± 90  54,000 ± 16000 >8,000 269 11000 ± 3000 850 ± 320 52000 ± 4000 >7,000 270  8000 ± 1500 1300 ± 100 >84,000  7100± 700 271 1700 ± 200 200 ± 90 >93,000  >9,300 272 >46,000  >4,700 >47,000  >4,700 273 30000 ± 5000  >1,500 >45,000  >4,500 27439000 ± 3000 1200 ± 300 >46,000  >4,500 275 1500 ± 300 370 ±40 >62,000  >6,200 276 1500 ± 200  760 ± 100 >61,000  >6,100 277 760 ±70 190 ± 20 31,000 ± 6000   9,800 ± 1000 278 1000 ± 100 270 ±10 >94000 >9,400 279 1700 ± 400 190 ± 20 >90000 >9,000 280 2400 ± 800   <80 >83000 >2,800 281 1800 ± 700 170 ± 10 27000 ± 2000 5000 ± 700 282680 ± 60 110 ± 10 >85000 >8,500 283  2900 ± 1200 300 ± 20 40000 ±4000 >9,300 284 13,600(N = 2) 340 ± 20 >8,800 285  3800 ± 1100 310 ± 2084000 ± 9000 2000 ± 100 286  48000 ± 10000 6300 ± 200 51000 ±1700 >8,800 287 455000 ± 22000 1100 ± 100 567000 ± 17000 4700 ± 400 2881800 ± 600 150 ± 20 11000 ± 3200 ~9,000 289 51 ± 4  530 ±150 >290000  >8,800 294  960 ± 170 295 200 ± 44 296  250 (N = 2) 297 2200 (N = 1) 298  >7000

What is claimed is:
 1. A composition comprising an Affinity MedicantConjugate (AMC) comprising an Affinity Moiety (AM) and a Medicant Moiety(MM), where the MM is an illudofulvene moiety selected from the groupconsisting of3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propanaloxime;2-(3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propylidene)hydrazine-1-carboxamide,6′-hydroxy-2′,4′,6′-trimethyl-3′-(3-(2-phenylhydrazineylidene)propyl)spiro[cyclopropane-1,5′-inden]-7′(6′H)-one,2-hydroxy-4-((R)-6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)butanenitrile,4-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)butanenitrile,3-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)propylsulfamate and1-(6′-hydroxy-2′,4′,6′-trimethyl-7′-oxo-6′,7′-dihydrospiro[cyclopropane-1,5′-inden]-3′-yl)methyl)pyrimidine-2,4(1H,3H)-dione,where the AM is selected from the group consisting of an antibody, achimeric antibody, a human antibody, a murine antibody, a humanizedantibody, a monoclonal antibody, an antibody fragment, a receptorprotein, a pro-peptide, a peptide, a protein, a glycopeptide, a proteasecleavable peptide, a growth factor, a steroid, a lipid, a liposomalparticle receptor binding species, an oligonucleotide and folate.
 2. Thecomposition further comprising the AMC of claim 1, and a physiologicallycompatible carrier.
 3. The composition further comprising the AMC ofclaim 1, in the form of a liposomal particle.
 4. The composition furthercomprising the AMC of claim 1, in the form of a nanoparticle.
 5. Thecomposition further comprising the AMC of claim 1, in the form of aPEGylated compound.
 6. The composition further comprising theR-enantiomers of the AMC of claim
 1. 7. The composition furthercomprising the S-enantiomers of the AMC of claim
 1. 8. The compositionfurther comprising a racemic mixture of the AMC of claim
 1. 9. Thecomposition of claim 1 forming a medicant.
 10. The composition furthercomprising the AMC of claim 1, where the AM is the antibody or antibodyfragment.
 11. The composition further comprising the AMC of claim 1,where the AM is the receptor protein.
 12. The composition furthercomprising the AMC of claim 1, where the AM is the growth factor. 13.The composition further comprising the AMC of claim 1, where the AM isthe lipid.
 14. The composition further comprising the AMC of claim 1,where the AM is the steroid.
 15. The composition further comprising theAMC of claim 1, where the AM is the protease cleavable peptide.
 16. Thecomposition further comprising the AMC of claim 1, where the AM is thepeptide or the glycopeptide.
 17. The composition further comprising theAMC of claim 1, where the AM is folate.
 18. The composition furthercomprising the AMC of claim 1, where the AM is the liposomal particlereceptor binding species.
 19. The composition further comprising the AMCof claim 1, where the AM is an anti-CD70 binding affinity moiety atoxin.
 20. The composition further comprising the AMC of claim 1, wherethe AM is the oligonucleotide.